阅读说明：本技术 用于快速检测核酸目标分子的存在的方法 (Method for rapid detection of the presence of nucleic acid target molecules ) 是由 弗拉迪米尔·哈吉恩 奈福斯·哈科 戴利博·哈科 姚祖栩 于 2018-12-27 设计创作，主要内容包括：一种室温隔板可储存的电泳阵列,用于在一溶液中快速检测多种预先选择的核酸目标分子中的至少一种核酸目标分子的存在的一方法,所述电泳阵列包括：多个固定的、相互隔开且相互电隔离的微凝胶沉积物,所述多个固定的、相互隔开且相互电隔离的微凝胶沉积物中的每一个都含有适于对所述多种预选的核酸目标分子中的至少一个进行滚环式扩增与结合的多种材料,所述多个微凝胶沉积物中的每一个都至少含有预先锚定在其中的下列元件：特异于所述多种预选核酸目标分子中的至少一种的一RCA探针；以及至少一个引物。(A room temperature partition storable electrophoretic array for use in a method of rapidly detecting the presence of at least one of a plurality of preselected nucleic acid target molecules in a solution, the electrophoretic array comprising: a plurality of immobilized, spaced apart and electrically isolated microgel deposits, each of said plurality of immobilized, spaced apart and electrically isolated microgel deposits containing a plurality of materials suitable for rolling-circle amplification and binding of at least one of said plurality of preselected nucleic acid target molecules, each of said plurality of microgel deposits containing at least the following elements pre-anchored therein: (ii) an RCA probe specific for at least one of the plurality of preselected nucleic acid target molecules; and at least one primer.)

1. The utility model provides an electrophoresis array that room temperature baffle can store which characterized in that: a method for rapidly detecting the presence of at least one of a plurality of preselected nucleic acid target molecules in a solution, said room temperature separator storable electrophoresis array comprising:

a plurality of immobilized, spaced apart and electrically isolated microgel deposits, each of said plurality of immobilized, spaced apart and electrically isolated microgel deposits containing a plurality of materials suitable for rolling-circle amplification and binding of at least one of said plurality of preselected nucleic acid target molecules, each of said plurality of microgel deposits containing at least the following elements pre-anchored therein:

(ii) an RCA probe specific for at least one of the plurality of preselected nucleic acid target molecules; and

at least one primer.

2. The electrophoretic array of claim 1, wherein: the plurality of microgel deposits are dehydrated and rehydratable when exposed to a solution containing at least one nucleic acid target molecule.

3. An electrophoretic array as claimed in claim 1 or 2, wherein: the at least one primer includes at least one forward primer and at least one reverse primer.

4. An electrophoretic array as claimed in any one of claims 1 to 3, wherein: the RCA probe is pre-hybridized to the at least one primer.

5. Electrophoretic array according to any of claims 1 to 4, wherein: when hydrated, each of the plurality of microgel deposits has a generally hemispherical configuration.

6. Electrophoretic array according to any of claims 1 to 5, wherein: the plurality of fixed, spaced apart and electrically isolated microgel deposits defining a corresponding plurality of fixed, spaced apart and electrically isolated microgel zones; and the electrophoretic array is used to implement a method comprising:

introducing the solution into each of the plurality of fixed, spaced apart and electrically isolated microgel zones;

performing at least substantially simultaneously rolling-ring amplification on each of the plurality of immobilized, mutually spaced and electrically isolated microgel regions while applying a plurality of electric fields to the plurality of microgel regions during respective stages of the rolling-ring amplification; and

detecting the presence of at least one of said plurality of preselected nucleic acid target molecules on at least one corresponding region of said plurality of immobilized, mutually spaced and electrically isolated microgels, wherein said detecting occurs within a short time of said introducing, said short time being less than 30 minutes.

7. The electrophoretic array of claim 6, wherein: the detecting comprises optical detecting.

8. The electrophoretic array of claim 6, wherein: the detection includes fluorescence detection.

9. Electrophoretic array according to any of claims 6 to 8, wherein: the applying the plurality of electric fields occurs at least two different stages in the rolling-circle amplification.

10. Electrophoretic array according to any of claims 6 to 9, wherein: the plurality of electric fields are at least substantially the same across each of the plurality of microgel zones that are fixed, spaced apart from each other, and electrically isolated from each other.

11. Electrophoretic array according to any of claims 6 to 10, wherein: the detecting occurs over a duration of less than 20 minutes.

12. Electrophoretic array according to any of claims 6 to 11, wherein: the detecting occurs over a duration of less than 15 minutes.

13. Electrophoretic array according to any of claims 6 to 12, wherein: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least one of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

14. Electrophoretic array according to any of claims 6 to 13, wherein: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least two of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of fixed, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

15. Electrophoretic array according to any of claims 6 to 14, wherein: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least three of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

16. Electrophoretic array according to any of claims 6 to 15, wherein: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least four of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

17. A method for extracting nucleic acid target molecules from a plurality of pre-selected nucleic acid target molecules in a solution and for rapidly detecting the presence of at least one of said nucleic acid target molecules, characterized by: the method comprises the following steps:

introducing the solution into at least a plurality of immobilized, spaced apart and electrically isolated microgel zones on an electrophoretic array, each of the plurality of immobilized, spaced apart and electrically isolated microgel zones containing a microgel deposit comprising a plurality of materials suitable for binding and rolling-circle amplification with different ones of the plurality of preselected nucleic acid target molecules;

performing at least substantially simultaneously rolling-circle amplification at the plurality of immobilized, mutually spaced and electrically isolated microgel regions, while applying a plurality of electric fields to the plurality of immobilized, mutually spaced and electrically isolated microgel regions during respective stages of the rolling-circle amplification; and

detecting the presence of at least one of the plurality of pre-selected nucleic acid target molecules and detecting at least one corresponding molecule in the plurality of immobilized, spaced apart and electrically isolated microgel regions;

wherein said detecting occurs within a short time of said introducing, said short time being less than 30 minutes.

18. The method for rapidly detecting the presence of at least one nucleic acid target molecule of claim 17, wherein: the detection comprises the following steps: and (4) optically detecting.

19. The method for rapidly detecting the presence of at least one nucleic acid target molecule of claim 17, wherein: the detection comprises the following steps: and (4) detecting fluorescence.

20. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 19, characterized in that: the applying the plurality of electric fields occurs in at least two stages of the rolling-circle amplification.

21. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 20, characterized in that: at each of the plurality of fixed, mutually spaced and electrically isolated microgel zones, the plurality of electric fields are at least substantially identical.

22. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 21, characterized in that: the detecting occurs over a duration of less than 20 minutes.

23. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 22, characterized in that: the detecting occurs over a duration of less than 15 minutes.

24. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 23, characterized in that: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least one of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

25. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 24, characterized in that: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least two of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

26. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 25, characterized in that: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least three of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

27. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 26, characterized in that: during the rolling-circle amplification, the applying a plurality of electric fields comprises at least four of:

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits;

applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and

applying an electric field of sequentially reversed polarity to the plurality of immobilized, spaced apart and electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

28. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 27, characterized in that: the electrophoretic array comprises an electrophoretic array storable in a room temperature partition.

29. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 17 to 28, characterized in that: the electrophoretic array includes:

a plurality of immobilized, spaced apart and electrically isolated microgel deposits, each of said plurality of immobilized, spaced apart and electrically isolated microgel deposits containing a plurality of materials suitable for rolling-circle amplification and binding of at least one of said plurality of preselected nucleic acid target molecules, each of said plurality of microgel deposits containing at least the following elements pre-anchored therein:

(ii) an RCA probe specific for at least one of the plurality of preselected nucleic acid target molecules; and at least one primer.

30. The method for rapidly detecting the presence of at least one nucleic acid target molecule of claim 29, wherein: the plurality of microgel deposits are dehydrated and rehydratable when exposed to a solution containing at least one nucleic acid target molecule.

31. The method for rapid detection of the presence of at least one nucleic acid target molecule according to claim 29 or 30, wherein: the at least one primer includes at least one forward primer and at least one reverse primer.

32. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 29 to 31, characterized in that: the RCA probe is pre-hybridized to the at least one primer.

33. The method for rapid detection of the presence of at least one nucleic acid target molecule according to any one of claims 29 to 32, characterized in that: when hydrated, each of the plurality of microgel deposits has a generally hemispherical configuration.

Technical field and Prior Art

The present invention relates to rolling circle amplification.

Disclosure of Invention

The present invention aims to provide various improved methods of rolling-circle amplification (RCA).

According to a preferred embodiment of the present invention, there is provided a room temperature partition storable electrophoretic array for use in a method of rapidly detecting the presence of at least one of a plurality of preselected nucleic acid target molecules in a solution, the electrophoretic array comprising: a plurality of immobilized, spaced apart and electrically isolated microgel deposits, each of said plurality of immobilized, spaced apart and electrically isolated microgel deposits containing a plurality of materials suitable for rolling-circle amplification and binding of at least one of said plurality of preselected nucleic acid target molecules, each of said plurality of microgel deposits containing at least the following elements pre-anchored therein: (ii) an RCA probe specific for at least one of the plurality of preselected nucleic acid target molecules; and at least one primer.

Preferably, the plurality of microgel deposits are dehydrated and rehydratable when exposed to a solution containing at least one nucleic acid target molecule. Additionally or alternatively, the at least one primer comprises at least one forward primer and at least one reverse primer.

According to a preferred embodiment of the invention, the RCA probe is pre-hybridized to the at least one primer.

According to a preferred embodiment of the present invention, each of the plurality of microgel deposits, when hydrated, has a generally hemispherical configuration.

According to a preferred embodiment of the present invention, the plurality of fixed, spaced apart and electrically isolated microgel deposits define a corresponding plurality of fixed, spaced apart and electrically isolated microgel zones; and the electrophoretic array is used to implement a method comprising: introducing the solution into each of the plurality of fixed, spaced apart and electrically isolated microgel zones; performing at least substantially simultaneously rolling-ring amplification on each of the plurality of immobilized, mutually spaced and electrically isolated microgel regions while applying a plurality of electric fields to the plurality of microgel regions during respective stages of the rolling-ring amplification; and detecting the presence of at least one of the plurality of preselected nucleic acid target molecules on at least one corresponding plurality of immobilized, mutually spaced and electrically isolated microgel zones, wherein said detecting occurs within a short time of said introducing, said short time being less than 30 minutes.

According to another preferred embodiment of the present invention, there is provided a method for rapidly detecting the presence of at least one nucleic acid target molecule among a plurality of pre-selected nucleic acid target molecules in a solution, the method comprising: introducing the solution into at least a plurality of immobilized, spaced apart and electrically isolated microgel zones on an electrophoretic array, each of the plurality of immobilized, spaced apart and electrically isolated microgel zones containing a microgel deposit comprising a plurality of materials suitable for binding and rolling-circle amplification with different ones of the plurality of preselected nucleic acid target molecules; performing at least substantially simultaneously rolling-circle amplification at the plurality of immobilized, mutually spaced and electrically isolated microgel regions, while applying a plurality of electric fields to the plurality of immobilized, mutually spaced and electrically isolated microgel regions during respective stages of the rolling-circle amplification; and detecting the presence of at least one of the plurality of pre-selected nucleic acid target molecules and detecting at least one corresponding molecule in the plurality of immobilized, spaced apart and electrically isolated microgel regions; wherein said detecting occurs within a short time of said introducing, said short time being less than 30 minutes.

According to a preferred embodiment of the invention, the detection comprises optical detection. Preferably, the detection comprises fluorescence detection.

According to a preferred embodiment of the present invention, said applying a plurality of electric fields occurs at least at two different stages in said rolling-circle amplification.

Preferably, the plurality of electric fields are at least substantially the same across each of the plurality of microgel zones that are fixed, spaced apart and electrically isolated from each other.

According to a preferred embodiment of the invention, said detection takes place over a duration of less than 20 minutes. More preferably, the detecting occurs over a duration of less than 15 minutes.

According to a preferred embodiment of the present invention, during the rolling-circle amplification, the applying the plurality of electric fields comprises at least one of the following steps: applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to drive a plurality of nucleic acid target molecules in the solution at the plurality of microgel deposits; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive products of hybridization of a plurality of nucleic acid target molecules in the solution with RCA probes to the plurality of microgel deposits; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to recapture RCA amplicons floating off the plurality of microgel deposits; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to drive a plurality of RCA probes into the plurality of microgel deposits to hybridize with at least one of a plurality of capture probes and a plurality of primers that have bound to the plurality of microgel deposits; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to remove unwanted molecules from the plurality of microgel zones; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to elongate RCA amplicons bound to the plurality of microgel deposits; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to compress RCA amplicons bound to the plurality of microgel deposits; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel regions to agitate a plurality of RCA reagents in the vicinity of RCA amplicons bound to the plurality of microgel deposits; applying an electric field to the plurality of immobilized, spaced apart and electrically isolated microgel zones to increase the rate of enzymatic activity in the RCA; and applying an electric field of sequentially reversed polarity to the plurality of immobilized, mutually spaced and mutually electrically isolated microgel regions to enhance the stringency of binding of the plurality of RCA amplicons to the plurality of microgel deposits.

According to another preferred embodiment of the present invention, the electrophoretic array comprises an electrophoretic array storable in a room temperature partition.

Preferably, the electrophoretic array comprises: a plurality of immobilized, spaced apart and electrically isolated microgel deposits, each of said plurality of immobilized, spaced apart and electrically isolated microgel deposits containing a plurality of materials suitable for rolling-circle amplification and binding of at least one of said plurality of preselected nucleic acid target molecules, each of said plurality of microgel deposits containing at least the following elements pre-anchored therein: an RCA probe specific for at least one of the plurality of preselected nucleic acid target molecules and at least one primer.

Description of the drawings

The invention will be more fully understood and appreciated from the following detailed description taken in conjunction with the drawings, in which:

FIGS. 1A and 1B are simplified assembly and exploded views of the construction and operation of components of an electrophoretic array comprising a plurality of immobilized, spaced apart and electrically isolated microgel regions during the operation of Rolling Circle Amplification (RCA), in accordance with a preferred embodiment of the present invention;

fig. 2A and 2B are simplified assembly and exploded views of the electrophoretic array assembly of fig. 1A and 1B, the electrophoretic array comprising: a plurality of fixed, spaced and electrically isolated microgel zones in an operational orientation for dehydrated storage;

FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H and 3I are simplified illustrations of a preferred method for fabricating the electrophoretic array employed in the embodiments of FIGS. 1A-2B;

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 4I and 4J are simplified pictorial illustrations of exemplary steps for rapidly detecting the presence of at least one nucleic acid target molecule, in accordance with an embodiment of the present invention;

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I and 5J are simplified pictorial illustrations of exemplary steps for rapidly detecting the presence of at least one nucleic acid target molecule, in accordance with an embodiment of the present invention;

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I and 6J are simplified diagrams of exemplary steps for rapidly detecting the presence of at least one nucleic acid target molecule, in accordance with an embodiment of the present invention;

FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H, FIG. 7I and FIG. 7J are simplified pictorial illustrations of exemplary steps for rapidly detecting the presence of at least one nucleic acid target molecule, in accordance with an embodiment of the present invention;

FIG. 8 is a graph summarizing the results of example I; and

fig. 9 is a graph summarizing the results of example II.

Detailed Description

Referring now to fig. 1A and 1B, fig. 1A and 1B are simplified assembled and exploded views of an electrophoretic array assembly 100, constructed and operative according to a preferred embodiment of the present invention, the electrophoretic array assembly 100 defining a plurality of microgel regions of immobilized, spaced apart and electrically isolated target-specific molecules during operation of Rolling Circle Amplification (RCA); referring now to fig. 2A and 2B, fig. 2A and 2B are simplified assembled and exploded views of the electrophoresis array assembly of fig. 1A and 1B, wherein a plurality of fixed, spaced apart and electrically isolated microgel zones are shown in an operational orientation for dehydrated storage. It should be appreciated that the electrophoretic array assembly 100 is particularly suitable for use in a method described below with reference to fig. 7A-7I.

As shown in fig. 1A and 1B, the electrophoresis array assembly 100 includes a housing defined by a substrate 110, a peripheral wall structure 120, and a window 130. Preferably, through a solution inlet hole 140 and a solution outlet hole 150 formed in the substrate 110 to enter the interior of the housing.

An electrophoretic array 160 is formed on the substrate 110, as will be described in more detail below with reference to fig. 3A-3I. The plurality of target molecule-specific microgel deposits 170 preferably have bound thereto a plurality of distinct RCA circular probes, a plurality of forward primers and a plurality of reverse primers, generally referred to herein by the reference numeral 175 in the form of a symbol, and immobilized on a plurality of separate working electrode locations 180, defined by the electrophoretic array 160. In fig. 1A and 1B, the plurality of target molecule-specific microgel deposits 170 are shown in an operational orientation suitable for rolling circle amplification.

In FIGS. 2A and 2B, the target molecule-specific microgel deposit is shown in a dehydrated state suitable for storage and is designated by reference numeral 190. It should be appreciated that the plurality of target molecule-specific microgel deposits 190 of fig. 2A and 2B, when hydrated, preferably assume the operational orientation shown in fig. 1A and 1B, which is indicated by reference numeral 170, by providing a suitable solution, preferably a solution containing nucleic acid target molecules, to the interior of the electrophoretic array assembly 100.

Various preferred dimensions of the electrophoresis array assembly 100 and its various components, for ease of calculation, assume that each microgel deposit 170 exposed to solution exhibits a generally hemispherical shape, as follows:

volume of solution: about 100 cubic millimeters.

Inner height between the substrate 110 and the window 130: 0.8 mm to 2.0 mm.

In the operational orientation of fig. 1A and 1B, the height of the plurality of target molecule-specific microgel deposits 170 above the substrate 110: 0.5 mm to 1.25 mm.

In the operational orientation of fig. 2A and 2B, the height of the plurality of target molecule-specific microgel deposits 190 above the substrate 110: 0.1 mm to 0.3 mm.

In the operational orientation of fig. 1A and 1B, the surface area of each target molecule-specific microgel deposit 170 above the substrate 110: 0.0016 to 0.009 square millimeters.

Ratio of surface area to solution volume of each target molecule-specific microgel deposit 170 exposed to the solution: there is an exposed surface area of the microgel deposit of 0.000016 to 0.00009 square millimeters per cubic millimeter of solution.

It should be understood that the actual surface area and the actual ratio of surface area in the volume of the solution is greater than or equal to that calculated via the simplifying assumption of a hemispherical shape.

The structure and construction of the electrophoretic array assembly 100 will now be described with reference to fig. 3A-3I.

Referring first to fig. 3A, it can be seen that the substrate 110 is typically a polyester sheet, such as: polyethylene terephthalate, preferably 0.1 mm thick. At this stage, the substrate 110 may be provided with a solution inlet hole 140 and a solution outlet hole 150, preferably by laser cutting. Alternatively, the solution inlet hole 140 and the solution outlet hole 150 may be formed in the substrate 110 at a later stage.

Referring now to fig. 3B, it can be seen that the substrate 110 is formed with an initial patterned layer 200 of a highly conductive material, preferably formed by screen printing of Henkel 479SS ink. Preferably, the thickness of layer 200 is 0.03 millimeters. Layer 200 provides a uniform electrical current conduction to each microgel deposit in the electrophoretic array assembly 100.

Fig. 3C shows a subsequently formed carbon layer 210, which is in registration with the initial patterned layer 200, and is preferably accomplished by screen printing dupont 7102 and BQ 221. Preferably, the thickness of layer 210 is 0.03 millimeters. Layer 210 serves as the working electrode material that is exposed to the solution, as described below. Layer 210 also controls the voltage applied between an inner working electrode 230 and an outer counter electrode 240.

The layers 200 and 210 in mutual registration together define an outer counter electrode 240 and an inner working electrode 230, the outer counter electrode 240 and the inner working electrode 230 being connected to a respective plurality of electrode contacts 250 and 260.

Referring now additionally to fig. 3D, it can be seen that a patterned dielectric layer 270 is formed over the plurality of layers 200 and 210 and over the substrate 100, preferably by screen printing of CMI-101-8079SS ink, which is available from Creative Materials, inc (Ayer, MA). Preferably, the dielectric layer 270 has a thickness of 0.04 mm.

As shown in fig. 3D, dielectric layer 270 is preferably formed with a plurality of holes 272 and 274, which holes 272 and 274 preferably correspond in size and location to solution inlet hole 140 and solution outlet hole 150. The dielectric layer 270 is also preferably provided with a plurality of elongated apertures 276 and 278 that cover portions of the opposing electrode 240. Each of the plurality of elongated apertures 276 and 278 preferably has a length of 100 millimeters. In addition, dielectric layer 270 is formed with a plurality of apertures 280, here shown as an array of eight apertures 280, that cover working electrode 230 and define locations 180 (fig. 1B and 2B) of separate working electrodes. Preferably, the plurality of holes 280 are all identical circular holes having a diameter of 0.2 mm to 0.5 mm, and a minimum spacing of 1 mm between the plurality of holes 280.

Referring now to fig. 3E, an array of microgel deposits 300 can be seen by mechanical spotting (spotting) on working electrode sites 180. Preferably, the plurality of microgel deposits 300 have a generally hemispherical shape when hydrated and a diameter of 0.70 mm in the plane of the dielectric layer 270 such that they are physically separated from each other. Preferably, the plurality of microgel deposits 300 have a height of 0.5 to 1.2 millimeters and an exposed surface area of 0.0016 to 0.009 square millimeters. Preferably, the plurality of microgel deposits 300 is formed from a (di) acrylamide hydrogel containing streptavidin.

Referring now to fig. 3F, preferably, it can be seen that the plurality of microgel deposits 300 are irradiated by ultraviolet rays to polymerize the components of the plurality of microgel deposits 300. Histidine is added to the plurality of microgel deposits 300, and then the plurality of microgel deposits 300 are washed to remove excess histidine.

After polymerization, the plurality of microgel deposits 300 are air dried to produce a plurality of dried microgel deposits 310, as shown in fig. 3G.

Referring now to fig. 3H, it can be seen that a plurality of different nucleic acid target molecule-specific RCA circular probes 320, and preferably a plurality of forward primers 322 and a plurality of reverse primers 324, are also bound to a corresponding different plurality of dried microgel deposits 310, e.g., by mechanical spotting, to produce different target molecule-specific microgel deposits 190 (fig. 2A and 2B). It will be appreciated that in the various figures, the plurality of nucleic acid target molecule-specific RCA circular probes 320, the plurality of forward primers 322 and the plurality of reverse primers 324 are indicated symbolically and are not shown to scale.

It should be understood that although the plurality of nucleic acid target molecule-specific RCA loop probes 320, the plurality of forward primers 322 and the plurality of reverse primers 324 are shown as being bound to the plurality of microgel deposits 310 as shown in the embodiments in fig. 3H to 3I and fig. 1A to 2B, in alternative embodiments, one or more of the plurality of nucleic acid target molecule-specific RCA loop probes 320, the plurality of forward primers 322 and the plurality of reverse primers 324 may not be bound to the plurality of microgel deposits 310 and may be provided in a solution with the plurality of nucleic acid target molecules. In the embodiments shown in fig. 3H-3I and in the alternative embodiments, the plurality of nucleic acid target molecule-specific RCA circular probes 320, the plurality of forward primers 322 and the plurality of reverse primers 324 are located in the immobilized, spaced apart and electrically isolated target molecule-specific microgel regions, as described below with reference to fig. 5A-7J.

After appropriate washing of the plurality of target molecule-specific microgel dried microgel deposits 190 and addition of raffinose (raffinose) as a preservative, the peripheral wall structure 120 and the window 130 are assembled onto the substrate 110, as shown in fig. 3I, thereby completing fabrication of the electrophoretic array assembly 100 in a partition storable state as described above with reference to fig. 2A and 2B. In accordance with a preferred embodiment of the present invention, the electrophoretic array assembly 100 is ready to provide a method for rapidly detecting the presence of at least one nucleic acid target molecule from a plurality of pre-selected nucleic acid target molecules, comprising, in one solution, the steps of:

introducing the solution into at least a plurality of immobilized, spaced apart and electrically isolated microgel zones on an electrophoretic array, each of the plurality of immobilized, spaced apart and electrically isolated microgel zones containing a microgel deposit comprising a plurality of materials suitable for binding and rolling-circle amplification with different ones of the plurality of preselected nucleic acid target molecules;

performing at least substantially simultaneously rolling-circle amplification at the plurality of immobilized, mutually spaced and electrically isolated microgel regions, while applying a plurality of electric fields to the plurality of immobilized, mutually spaced and electrically isolated microgel regions during respective stages of the rolling-circle amplification; and

detecting the presence of at least one of the plurality of pre-selected nucleic acid target molecules and detecting at least one corresponding molecule in the plurality of immobilized, spaced apart and electrically isolated microgel regions;

wherein said detection occurs within a short time of said introduction, preferably said short time is less than 30 minutes, more preferably said short time is less than 25 minutes, even more preferably said short time is less than 20 minutes.

In the following description, four variations of performing the above-described method are described in detail with reference to fig. 4A to 4J, fig. 5A to 5J, fig. 6A to 6J, and fig. 7A to 7J, respectively. The electrophoretic array assembly 100 described above is particularly suitable for performing the methods of fig. 7A-7J.

All of these methods employ rolling circle amplification. Rolling circle amplification is a known technique, described in particular in the following publications, the disclosures of which are incorporated herein by reference:

U.S. Pat. nos. 5,854,033; lizardi et al, Nature Genetics 19 (3): 225-232(1998).

Michael G.Mohsen and Eric T.Kool, Rolling Circle Amplification and Rolling Circle transformation, Acc Chem Res.2016,49(11): 2540-.

Identification and Typing of Isolates of cytopathora and related by Use of Amplified Fragment Length polymorphism and Rolling Circle Amplification of Peiying Feng et al, Journal of Clinical Microbiology, Volume 51 Number 3 in 2013, p.931-937.

Nucleic Acids Research, 2001, Vol.29, No.123, e118, by Signal Amplification by Rolling Circle Amplification on DNAmicroarray, G.Nallur et al.

M. Monsur Ali et al, Rolling circle amplification, a versatile tool for Chemical biology, materials science and media, Chemical Society Review, chem. Soc. Rev.,2014, 43,3324 and 3341.

Ali MM, Li F, Zhang Z, Zhang K, Kang D, Ankrum JA, Le XC, ZHao W.Rollingcircle amplification, a versatil tool for chemical biology, materials science and medicine. chem Soc Rev.2014; 43: 3324.

kool, ET. Rolling circle synthesis of oligonucleotides and amplification of selected random circular oligonucleotides, US.5714320.1998, 2/3.

Fire A, Xu S.Rolling reproduction of short DNA circuits, Proc Natl Acadsi U S.1995; 92: 4641-4645.

Nilsson M, Malmgren H, samitoki M, kwatatkowski M, chowdary BP, landegren u.padlock Probes: circulating Oligonucleotides for Localized DNAseDetection.science.1994; 265:2085-2088.

The various methods described below include a number of features that are novel and unobvious relative to prior art rolling circle amplification techniques.

Referring now to fig. 4A-4J, fig. 4A-4J illustrate major stages in rapid detection of the presence of at least one nucleic acid target molecule from a plurality of pre-selected nucleic acid target molecules, according to a preferred embodiment of the present invention.

Preferably, the method of fig. 4A-4J may be performed using the electrophoretic array assembly 100 in a partition storable state, as described above with reference to fig. 2A and 2B, in which only the plurality of capture probes are associated with the plurality of immobilized, spaced apart and electrically isolated microgel deposits 190. It should be understood that for simplicity, the substrate 110, the peripheral wall structure 120, and the window 130 and various layers that make up the electrophoretic array 160 are as described above with reference to fig. 1A-2B, and are not specifically shown in fig. 4A-4J. Each of fig. 4A-4J is a simplified side cross-sectional view taken generally along lines 4-4 of fig. 2A.

FIG. 4A shows the electrophoresis array assembly 100 in its partition storable state as shown in FIG. 2A, the electrophoresis array assembly 100 having a plurality of indications of various symbolic, not to scale, oligonucleotide nucleic acid specific capture probes 400, such as those specific for but not limited to identifying meningitis infectious disease pathogens or other diseases associated with a plurality of corresponding different dried microgel deposits 190. Fig. 4A shows the internal dimensions of a preferred embodiment of the electrophoretic array assembly 100 in millimeters, along with the general dimensions of the immobilized dried target molecule-specific microgel deposit 190, centered on a plurality of working electrode locations 180 but extending slightly (fig. 1A-3I).

FIG. 4B shows the introduction of a solution 402 containing one or more different types of nucleic acid target molecules 403, represented by an arrow 401, to fill the interior of the electrophoretic array assembly 100. Preferably, the solution 402 further comprises a plurality of nucleic acid target molecules 403, a plurality of forward primers 322 and a plurality of nucleic acid target molecule-specific RCA circular probes 320.

The preparation of the solution 402 is not part of the present patent application and is carried out according to conventional techniques, such as the techniques described in "Nasir Ali, Rita de C a Pontello rampczo, Alexandre dia Tavares Costa and marcoarelio Krieger, current methods of nucleic acid extraction and impact on instant diagnosis, biomedrearch International Volume 2017, topic No.: 9306564, page 13 ". Preferably, solution 402 includes a low conductivity eluent, typically introduced during preparation of the solution 402, that facilitates electronic addressing of the various nucleic acids and facilitates the activity of restriction enzymes in solution 402. A preferred eluent comprises histidine and a restriction enzyme buffer. Fig. 4B shows the dried target molecule-specific microgel deposit 190 in a dehydrated state. The solution 402 is introduced into the electrophoresis array assembly 100, the solution 402 is contacted with the dried target molecule-specific microgel deposit 190 for defining a time T0, and the dried target molecule-specific microgel deposit 190 is allowed to assume its hydrated state, designated by reference numeral 170 (fig. 1A and 1B).

Fig. 4C shows a plurality of immobilized target molecule-specific microgel deposits 170 in a monohydrate state as a result of introducing a solution 402 containing a plurality of nucleic acid target molecules 403, the nucleic acid target molecules 403 filling the interior of the electrophoretic array assembly 100. Fig. 4C shows the plurality of hydrated target molecule-specific electrically isolated microgel deposits 170 having a typical maximum height of 1.25 millimeters. Preferably, the hydration of the plurality of target molecule-specific microgel deposits 170 to the state shown in fig. 4C requires about 10 seconds, and is preferably completed at time T ═ T0+10 seconds.

FIG. 4D shows electrophoretic addressing of nucleic acids to the hydrated immobilized, mutually electrically isolated microgel deposits 170 between working electrode 230 and working electrode sites 180 on counter electrode 240 in the presence of an applied Direct Current (DC) electric field, preferably 10 to 300 volts/cm (FIGS. 1A and 1B). Portions of the plurality of electric field lines (electric fields) are indicated by reference numerals 410 and the direction of the electric field is indicated by arrows 412. It should be appreciated that the plurality of electric field lines define a plurality of three-dimensional, fixed, spaced apart and electrically isolated regions 420 of the target molecule-specific microgel, each surrounding a different target molecule-specific microgel deposit 170.

It should be understood that the addressing and the various steps described below with reference to fig. 4E-4J occur not only on the surface of the plurality of microgel deposits 170, but also within the volume of the plurality of microgel deposits 170.

As shown in fig. 4D, the application of the DC electric field to the three-dimensional, fixed, spaced apart and electrically isolated target molecule-specific microgel regions 420 results in rapid transport of a plurality of nucleic acid target molecules 403, a plurality of nucleic acid target molecule-specific RCA circular probes 320 and a plurality of forward primers 322 to the plurality of target molecule-specific microgel deposits 170, thereby promoting specific hybridization between the nucleic acid target molecule 403 and the nucleic acid target molecule-specific RCA circular probe 320, specific hybridization between the plurality of forward primers 322 and the plurality of nucleic acid target molecule-specific RCA circular probes 320, and capturing the plurality of nucleic acid target molecule-specific RCA circular probes 320 by a plurality of target molecule-specific capture probes 400, the plurality of target molecule-specific capture probes 400 are bound to the hydrated target molecule-specific electrically isolated microgel deposits 170.

The duration of the phase shown in fig. 4D is between 30 seconds and 120 seconds. Preferably, the phase shown in fig. 4D is completed at time T ═ T0+ [40 to 130] seconds.

Reference is now made to fig. 4E, which shows a connect phase generally following the address phase shown in fig. 4D, and preferably in the presence of a DC electric field, preferably 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 4D. Preferably, the duration of the connection phase is typically 120 to 240 seconds. Preferably, the connection phase is completed in T ═ T0+ [160 to 370] seconds.

Referring now to fig. 4F, there is shown an RCA polymerisation phase generally following the ligation phase shown in fig. 4E, and preferably occurring in the presence of a DC electric field, preferably 10 to 300 volts/cm in the same direction as the electric field of the step shown in fig. 4E.

Preferably, the RCA polymerisation stage takes place in the presence of a Bst polymerase 429, a plurality of dntps (not shown) and a reverse primer 324, which are introduced into the electrophoretic array assembly 100 via a solution, and the forward primer 322 is bound to the RCA circular probe 320, which in turn is bound to the capture probe 400, which in turn is bound to the target molecule specific microgel deposit 170, preferably at a temperature of 65 ℃. One result of the RCA polymerization stage is the production of long RCA amplicons 440. As shown in fig. 4F, after polymerase 429 binds to the plurality of nucleic acid target molecule-specific RCA circular probes 320, the plurality of nucleic acid target molecules 403 are displaced from the plurality of nucleic acid target molecule-specific RCA circular probes 320, as indicated by the plurality of arrows 442. The duration of the RCA polymerisation stage is typically 300 to 720 seconds. Preferably, the RCA polymerisation stage is completed in T ═ T0+ [460 to 1090] seconds.

Referring now to fig. 4G, there is shown an amplicon elongation phase that generally occurs during the RCA polymerization phase of fig. 4F, and preferably occurs in the presence of a DC electric field, preferably 10 to 300 volts/cm in a direction opposite the electric field in the step of fig. 4F, as indicated by arrow 444. In the illustrated embodiment, elongation of the amplicon 440 occurs in a direction indicated by an arrow 448. Preferably, the amplicon extension phase is performed in the presence of a Bst polymerase 429, dntps (not shown), and reverse primer 324 at a temperature of 65 ℃. The duration of the amplicon extension phase is typically 5 to 15 seconds. Preferably, the RCA polymerization stage is completed in T ═ T0+ [ 465-.

It should be understood that the stages shown in fig. 4F and 4G may be repeated intermittently a plurality of times, each stage of the plurality of stages shown in fig. 4F being shorter in duration than described above and separated by a stage shown in fig. 4G.

Referring now to fig. 4H, there is shown an exponential RCA amplification phase that occurs generally during the RCA polymerization phase of fig. 4F and the amplicon elongation phase of fig. 4G, and preferably in the presence of a DC electric field, preferably 10 to 300 volts/cm in a direction opposite the electric field in the step of fig. 4G, as indicated by arrow 412, but preferably including short periods of reversal of the electric field polarity. At this stage, a plurality of additional amplicons 450 are generated using the reverse primer 324.

Preferably, the exponential RCA amplification stage occurs in the presence of a Bst polymerase 429, a plurality of dntps (not shown), and a reverse primer 324 at a temperature of 65 ℃. The duration of the exponential RCA amplification phase occurring during the RCA polymerization phase of fig. 4F and the amplicon extension phase of fig. 4G is typically 5 to 15 seconds. The RCA polymerization phase of fig. 4F, the amplicon extension phase of fig. 4G, and the exponential RCA amplification phase of fig. 4H are preferably completed in T ═ T0+ [465 to 1105] seconds.

Referring now to fig. 4I, it is shown that a post-RCA polymerization addressing phase, preferably 10 to 300 volts/cm in the presence of a DC electric field, preferably in the same one direction as the electric field in the step of fig. 4H, typically occurs after completion of the RCA polymerization phase of fig. 4F, the amplicon extension phase of fig. 4G, and the exponential RCA amplification phase of fig. 4H. The post-RCA polymerization addressing stage is particularly helpful for concentrating the amplicons 440, which amplicons 440 are in solution 402 at a distance from the plurality of target molecule-specific microgel deposits 170 and recaptured at the target molecule-specific microgel deposits 170, as indicated by arrows 460, preferably aggregating the plurality of amplicons 440 at one location within each microgel region 420. The duration of the post-RCA polymerization addressing phase is typically 10 seconds to 30 seconds. Preferably, the post-RCA polymerisation addressing stage is completed in T ═ T0+ [475 to 1135] seconds.

Referring now to fig. 4J, there is shown a reporting phase generally following the post RCA aggregation addressing phase. Preferably, the reporting phase occurs in the presence of fluorescent reporters 470 complementary to the amplicons 440, 450, which amplicons 440, 450 are introduced into the electrophoretic array assembly 100 through a solution. The duration of the reporting phase is typically 10 to 30 seconds. Preferably, the reporting phase is completed in T ═ T0+ [485 to 1165] seconds.

After completion of the reporting phase and a subsequent washing phase (not shown), the presence of at least one nucleic acid target molecule can be detected from a plurality of pre-selected nucleic acid target molecules via conventional fluorescence detection. Thus, it should be understood that preferably the detection of at least one nucleic acid target molecule should be completed within 8 minutes to 20 minutes of the initial supply of solution 402 to the interior of the electrophoretic array assembly 100.

It will be appreciated that if the preparation of the solution 402 is completed within 4 minutes to 5 minutes of obtaining a sample (e.g.by a blood sample taken from a patient), the at least one nucleic acid target molecule may be detected from the sample within 12 minutes to 25 minutes.

Referring now to fig. 5A-5J, fig. 5A-5J illustrate major stages in rapid detection of the presence of at least one nucleic acid target molecule from a plurality of pre-selected nucleic acid target molecules, according to another preferred embodiment of the present invention.

Preferably, the method of fig. 5A-5J is performed using an electrophoretic array assembly 500 in a partition storable state, as described above with reference to fig. 2A and 2B, wherein the plurality of forward primers 322 are combined with the plurality of immobilized and spaced apart microgel deposits 190. It should be understood that the substrate 110, the peripheral wall structure 120, and the window 130 and the various layers making up the electrophoretic array 160 are not specifically shown for the sake of brevity. Each of fig. 5A-5J is a simplified side cross-sectional view taken generally along line 4-4 in fig. 2A.

Fig. 5A shows an electrophoretic array assembly 500 similar to that shown in fig. 2A in a dried, dehydrated, operational orientation, the electrophoretic array assembly 500 having multiple indications of various oligonucleotide forward primers 322, which are not drawn to scale, and which bind to corresponding different ones of the multiple dried microgel deposits 190.

Fig. 5A shows the internal dimensions of a preferred embodiment of the electrophoresis array assembly 500 in millimeters, along with the general dimensions of the plurality of immobilized dried target molecule-specific microgel deposits 190.

It should be understood that the method of fig. 5A to 5J is different from the method of fig. 4A to 4J in that, instead of the plurality of capture probes 400 being combined with the plurality of immobilized dried target molecule-specific microgel deposits 190 in the method of fig. 4A to 4J, the plurality of forward primers 322 are also used as the plurality of capture probes and combined with the plurality of immobilized dried target molecule-specific microgel deposits 190 in the method of fig. 5A to 5J.

FIG. 5B shows the introduction of a solution 502 containing a plurality of nucleic acid target molecules 503, represented by an arrow 501, to fill the interior of the electrophoretic array assembly 500. The solution preferably comprises a plurality of nucleic acid target molecule-specific RCA loop probes 320 in addition to the plurality of nucleic acid target molecules 503.

The preparation of the solution 502 is not part of the claimed invention and is carried out according to conventional techniques, such as those described in "Nasir Ali, ritac a pintello rampazzo, Alexandre Dias Tavares Costa and marcoarelio Krieger, Current Nucleic Acid, extraction methods and their effect on instant diagnosis, BioMed Research International, 2017, article number: 9306564, page 13 ". Preferably, solution 502 includes a low conductivity eluent, typically introduced during preparation of the solution, that facilitates electronic addressing of nucleic acids and facilitates activity of restriction enzymes in solution 502. A preferred eluent comprises histidine and a restriction enzyme buffer. Fig. 5B shows a plurality of dried target molecule-specific microgel deposits 190 in a dehydrated state. Solution 502 is introduced into electrophoresis array assembly 500 such that solution 502 contacts dried target molecule-specific microgel deposits 190, defines a time T0 and hydrates dried target molecule-specific microgel deposits 190 (fig. 2A and 2B) to their hydrated form, indicated by reference numeral 170 (fig. 1A and 1B).

Fig. 5C shows a plurality of immobilized target molecule-specific microgel deposits 170 in a monohydrate state, and as a result of introducing a solution 502 containing a plurality of nucleic acid target molecules 503, the plurality of nucleic acid target molecules 503 fill the interior of the electrophoretic array assembly 500. Fig. 5C shows the plurality of hydrated target molecule-specific electrically isolated microgel deposits 170 having a typical maximum height of 1.25 millimeters. The hydration of the plurality of molecular-specific microgel deposits 170 to the state shown in fig. 5C preferably takes about 10 seconds, and is preferably completed at time T ═ T0+10 seconds.

FIG. 5D shows electrophoretically addressing nucleic acids to a plurality of hydrated, immobilized, mutually electrically isolated deposits 170 of target molecule-specific microgels in the presence of a DC electric field (preferably 10 volts to 300 volts/cm). Portions of the plurality of electric field lines are represented by reference numeral 510, and the direction of the electric field is represented by a plurality of arrows 512. It should be appreciated that the electric field lines define a plurality of three-dimensional, stationary, spaced apart and electrically isolated microgel zones 520, each centered around a different target molecule-specific microgel deposit 170.

It should be understood that addressing and the various steps described below with reference to fig. 5E through 5J occur not only on the surface of the microgel deposit 170, but also within the volume of the plurality of microgel deposits 170.

As shown in fig. 5D, application of the DC electric field to the plurality of three-dimensional, immobilized, spaced apart and electrically isolated target molecule-specific microgel regions 520 results in rapid transport of the plurality of nucleic acid target molecules 503 and the plurality of nucleic acid target molecule-specific RCA circular probes 320 to the plurality of target molecule-specific microgel deposits 170, thereby promoting specific hybridization between the plurality of nucleic acid target molecules 503 and the plurality of nucleic acid target molecule-specific RCA circular probes 320. Rapid transport of a plurality of nucleic acid target molecule-specific RCA circular probes 320 to the plurality of target molecule-specific microgel deposits 170 also promotes specific hybridization between the plurality of forward primers 322, which plurality of forward primers 322 bind to the plurality of target molecule-specific microgel deposits 170 and the plurality of nucleic acid target molecule-specific RCA circular probes 320.

The duration of the phase shown in fig. 5D is between 30 seconds and 120 seconds. Preferably, the phase shown in fig. 5D is completed in a time T ═ T0+ [40 to 130] seconds.

Reference is now made to fig. 5E, which illustrates a connect phase generally following the addressing phases shown in fig. 5D, and preferably in the presence of a DC electric field, preferably 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 5D. Preferably, the ligation phase occurs in the presence of a ligase 528 (e.g., T-4 ligase), which ligase 528 is introduced into the electrophoretic array assembly 500 via a solution. The duration of the connection phase is typically 120 to 240 seconds. Preferably, the connection phase is completed in T ═ T0+ [160 to 370] seconds.

Referring now to fig. 5F, there is shown an RCA polymerisation phase generally following the ligation phase shown in fig. 5E, and preferably occurring in the presence of a DC electric field, preferably 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 5E. Preferably, the RCA polymerization stage occurs in the presence of a Bst polymerase 529, dntps (not shown), and a reverse primer 324, the reverse primer 324 being introduced into the electrophoretic array assembly 500 via solution, and a forward primer 322 binding to the target molecule binding specific microgel deposit 170, preferably at 65 ℃. One result of the RCA polymerization stage is the generation of multiple long RCA amplicons 540 (fig. 5G). As shown in fig. 5F, after polymerase 529 binds to the plurality of nucleic acid target molecule-specific RCA loop probes 320, the plurality of nucleic acid target molecules 503 are displaced from the plurality of nucleic acid target molecule-specific RCA loop probes 320, as indicated by arrows 542. The duration of the RCA polymerisation stage is typically 300 to 720 seconds. Preferably, the RCA polymerisation stage is completed in T ═ T0+ [560 to 1090] seconds.

Referring now to fig. 5G, there is shown an amplicon elongation phase that generally occurs during the RCA polymerization phase of fig. 5F, and preferably occurs in the presence of a DC electric field, preferably 10 to 300 volts/cm in a direction opposite the electric field in the step of fig. 5F, as indicated by an arrow 544. The elongation of the amplicon 540 occurs in a direction indicated by an arrow 548. Preferably, the amplicon elongation phase occurs in the presence of a Bst polymerase 529, dntps (not shown), and reverse primers 324 at a temperature of 65 ℃. The duration of the amplicon extension phase is typically 5 to 15 seconds. Preferably, the RCA polymerization stage is completed in T ═ T0+ [ 565-.

It should be understood that the various stages shown in fig. 5F and 5G may be repeated intermittently a plurality of times, each of the plurality of stages shown in fig. 5F being shorter in duration than described above and being separated by a stage shown in fig. 5G.

Referring now to fig. 5H, there is illustrated an exponential RCA amplification phase that generally occurs during the RCA polymerization phase of fig. 5F and the amplicon extension phase of fig. 5G, and preferably occurs in the presence of a DC electric field, preferably 10 to 300 volts/cm in a direction opposite the electric field in the step of fig. 5G, as shown by arrows 512, but preferably including short periods of reversal of the electric field polarity. At this stage, a plurality of reverse primers 324 are used to generate a plurality of additional amplicons 550.

Preferably, the exponential RCA polymerization phase occurs in the presence of a Bst polymerase 529, dntps (not shown), and a reverse primer 324 at a temperature of 65 ℃, the reverse primer 324 being introduced into the electrophoretic array assembly 500 via solution, and a forward primer 322 preferably binding to the target molecule binding specific microgel deposit 170 at 65 ℃. The duration of the exponential RCA amplification phase occurring during the RCA polymerization phase of fig. 5F and the amplicon extension phase of fig. 5G is typically 5 to 15 seconds. Preferably, the RCA polymerization phase of fig. 5F, the amplicon extension phase of fig. 5G, and the exponential RCA amplification phase of fig. 5H are completed in T ═ T0+ [ 465-.

Reference is now made to fig. 5I, which illustrates that a post-RCA polymerization addressing phase typically occurs after completion of the RCA polymerization phase of fig. 5F, the amplicon extension phase of fig. 5G, and the exponential RCA amplification phase of fig. 5H, and is preferably 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 5H. The post-RCA polymerization addressing stage is particularly useful for collecting the plurality of amplicons 540 located in the solution 502, the plurality of amplicons 540 being at a distance from and recapturing the plurality of target molecule-specific microgel deposits 170, as indicated by the plurality of arrows 560, preferably concentrating the plurality of amplicons 540 at a location within each microgel region 520. The duration of the post-RCA polymerization addressing phase is typically 10 seconds to 30 seconds. Preferably, the post-RCA polymerisation addressing stage is completed in T ═ T0+ [475 to 1135] seconds.

Referring now to fig. 5J, there is shown a reporting phase generally following the post-RCA polymerization addressing phase. Preferably, the reporting phase occurs in the presence of a fluorescent reporter 470 complementary to the amplicons 540, 550, which are introduced into the electrophoretic array assembly 500 through a solution. The duration of the reporting phase is typically 10 to 30 seconds. Preferably, the reporting phase is completed in T ═ T0+ [585 to 1165] seconds.

After completion of the reporting phase and a subsequent washing phase (not shown), the presence of at least one nucleic acid target molecule can be detected from a plurality of pre-selected nucleic acid target molecules via conventional fluorescence detection. Thus, it should be understood that preferably, the detection of at least one nucleic acid target molecule should be completed within 8 minutes to 20 minutes of the initial supply of solution 502 to the interior of the electrophoretic array assembly 100.

It is understood that if the preparation of solution 502 is completed within 4 minutes to 5 minutes of obtaining a sample (e.g., by a blood sample taken from a patient), at least one nucleic acid target molecule may be detected from the sample within 12 minutes to 25 minutes.

Referring now to fig. 6A-6J, fig. 6A-6J illustrate major stages in rapid detection of the presence of at least one nucleic acid target molecule from a plurality of pre-selected nucleic acid target molecules, according to another preferred embodiment of the present invention.

Preferably, the method of fig. 6A-6J may be performed using the electrophoretic array assembly 600 in a separator storable state, as described above with reference to fig. 2A and 2B, wherein the plurality of forward primers 322 and the plurality of nucleic acid target molecule-specific RCA loop probes 320 are bound to the plurality of immobilized, mutually spaced apart microgel deposits 190. It should be understood that the substrate 110, the peripheral wall structure 120, and the window 130, as well as the various layers making up the electrophoretic array 160 are not specifically shown for the sake of brevity. Each of fig. 6A-6J is a simplified side cross-sectional view taken generally along lines 4-4 of fig. 2A.

Fig. 6A shows an electrophoretic array assembly 500 similar to that shown in fig. 2A in a dry, dehydrated, operational orientation, the electrophoretic array assembly 500 having indications of a symbolic, not to scale, variety of different oligonucleotide forward primers 322 and a plurality of nucleic acid target molecule-specific RCA loop probes 320, the plurality of nucleic acid target molecule-specific RCA loop probes 320 being bound to a corresponding plurality of different dry microgel deposits 190.

Fig. 6A shows the internal dimensions of a preferred embodiment of the electrophoresis array assembly 600 in millimeters, along with the general dimensions of the plurality of immobilized dried target molecule-specific microgel deposits 190.

It should be understood that the method of fig. 6A to 6J differs from the method of fig. 4A to 4J in that, instead of the plurality of capture probes 400 being bound to the plurality of immobilized dried target molecule-specific microgel deposits 190 in the method of fig. 4A to 4J, a plurality of nucleic acid target-specific RCA probes 320 are specifically bound to a plurality of forward primers 322 are also bound to the plurality of immobilized dried target molecule-specific microgel deposits 190 in the method of fig. 6A to 6J.

FIG. 6B shows the introduction of a solution 602 containing a plurality of nucleic acid target molecules 603, represented by an arrow 601, to fill the interior of the electrophoretic array assembly 600.

The preparation of the solution 602 is not part of the claimed invention and the invention is carried out according to conventional techniques, such as "Nasir Ali, Rita de C a sia Pontello Rampazzo, Alexandre Dias Tavares Costa and Marco Aureliao Kriger, Current Nucleic Acid Extraction Methods and therapeutics to Point-of-Care Diagnostics, BioMed Research International, 2017, article number 93065". Preferably, solution 602 typically includes a low conductivity eluent introduced during the preparation of the solution that facilitates electronic addressing of nucleic acids and promotes the activity of restriction enzymes in solution 602. A preferred eluent comprises histidine and a restriction enzyme buffer. Fig. 6B shows the plurality of dried target molecule-specific microgel deposits 190 in a dehydrated state. The solution 602 is introduced into the electrophoresis array assembly 100 such that the solution 602 contacts the plurality of dried target molecule-specific microgel deposits 190, defines a time T0 and causes the plurality of dried target molecule-specific microgel deposits 190 to assume their hydrated state, as indicated by reference numeral 170 (fig. 1A, fig. 1B).

Fig. 6C shows a plurality of immobilized target molecule-specific microgel deposits 170 in a monohydrate state as a result of introducing a solution 602 containing a plurality of nucleic acid target molecules 603, and filling the interior of the electrophoretic array assembly 600. Fig. 6C shows the plurality of target molecule-specific electrically isolated microgel deposits 170 hydrated, the plurality of microgel deposits 170 having a typical maximum height of 1.25 millimeters. Preferably, it takes about 10 seconds for the plurality of target molecule-specific microgel deposits 190 to hydrate to the state shown in fig. 6C, and is preferably completed at time T ═ T0+10 seconds.

Preferably, FIG. 6D shows electrophoretic addressing of nucleic acids to a plurality of hydrated, immobilized, mutually electrically isolated deposits 170 of target molecule-specific microgels in the presence of a DC electric field of 10 volts to 300 volts/cm. Portions of the plurality of electric field lines are represented by a plurality of reference numerals 610, and the direction of the electric field is represented by a plurality of arrows 612. It should be appreciated that the plurality of electric field lines define a plurality of three-dimensional, fixed, spaced apart and electrically isolated microgel zones 620. Each microgel region 620 surrounds a different target molecule specific microgel deposit 170.

It should be understood that addressing and the various steps described below with reference to fig. 6E-6J occur not only on the surface of the plurality of microgel deposits 170, but also within the volume of the plurality of microgel deposits 170.

As shown in fig. 6D, application of the DC electric field to the plurality of three-dimensional, immobilized, spaced apart and electrically isolated target molecule-specific microgel regions 620 results in rapid transport of a plurality of nucleic acid target molecules 603 to the plurality of target molecule-specific microgel deposits 170, thereby promoting specific hybridization between the plurality of nucleic acid target molecules 603 and the plurality of nucleic acid target molecule-specific RCA loop probes 320, wherein the plurality of RCA loop probes 320 are bound to a plurality of forward primers 322, wherein the plurality of forward primers 322 are bound to the hydrated plurality of target molecule-specific electrically isolated microgel deposits 170.

The duration of the phase shown in fig. 6D is between 30 and 120 seconds. Preferably, the phase shown in fig. 6D is completed at time T ═ T0+ [40 to 130] seconds.

Referring now to fig. 6E, there is shown a connect phase generally following the address phase shown in fig. 6D, preferably in the presence of a DC electric field, preferably 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 6D. Preferably, the ligation phase occurs in the presence of a ligase 628 (e.g., T-4 ligase), which is introduced into the electrophoretic array assembly 600 via a solution. The duration of the connection phase is typically 120 to 240 seconds. Preferably, the connection phase is completed in T ═ T0+ [160 to 370] seconds.

Reference is now made to fig. 6F, which illustrates a RCA polymerisation phase generally following the ligation phase shown in fig. 6E, and preferably occurring in the presence of a DC electric field, preferably 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 6E. Preferably, the RCA polymerization stage takes place in the presence of a Bst polymerase 629, dntps (not shown) and a reverse primer 324 (the reverse primer 324 is introduced into the electrophoretic array assembly 600 by solution) and a forward primer 322 (the forward primer 322 binds to the target molecule specific microgel deposit), and preferably at a temperature of 65 ℃. One result of the RCA polymerization stage is the generation of multiple long RCA amplicons 640 (fig. 6G). As shown in fig. 6F, after polymerase 629 binds to the plurality of nucleic acid target molecule-specific RCA loop probes 320, the plurality of nucleic acid target molecules 603 are displaced from the plurality of nucleic acid target molecule-specific RCA loop probes 320, as shown by the plurality of arrows 642. The duration of the RCA polymerisation stage is typically 300 to 720 seconds. Preferably, the RCA polymerisation stage is completed in T ═ T0+ [460 to 1090] seconds.

Referring now to fig. 6G, there is shown a stage of amplicon elongation that typically occurs during the RCA polymerization stage of fig. 6F, and preferably occurs in the presence of a DC electric field, preferably 10 to 300 volts/cm in a direction opposite the electric field in the step of fig. 6F. The elongation of the amplicon 640 occurs in a direction indicated by an arrow 648. Preferably, the RCA polymerization stage occurs in the presence of a Bst polymerase 629, dntps (not shown), and a reverse primer 324 at a temperature of 65 ℃. The duration of the amplicon extension phase is typically 5 to 15 seconds. Preferably, the RCA polymerization stage is completed in T ═ T0+ [ 465-.

It should be understood that the stages shown in fig. 6F and 6G include: the multiple stages shown in fig. 6F may be repeated intermittently a plurality of times, each of the multiple stages shown in fig. 6F having a duration that is shorter than the time described above and separated by a stage shown in fig. 6G.

Referring now to fig. 6H, there is shown an exponential RCA amplification phase that occurs generally during the RCA polymerization phase of fig. 6F and the amplicon extension phase of fig. 6G, and preferably in the presence of a DC electric field, preferably 10 to 300 volts/cm in a direction opposite the electric field in the step of fig. 6G, but preferably including short periods of electric field polarity reversal. At this stage, an additional plurality of amplicons 650 are generated using the plurality of reverse primers 324.

Preferably, the exponential RCA amplification stage occurs in the presence of a Bst polymerase 629, a plurality of dntps (not shown), and a reverse primer 324 at a temperature of 65 ℃. The duration of the exponential RCA amplification phase occurring during the RCA polymerization phase of fig. 6F and the amplicon extension phase of fig. 6G is typically 5 to 15 seconds. Preferably, the RCA polymerization stage of fig. 6F, the amplicon extension stage of fig. 6G, and the exponential RCA amplification stage of fig. 6H are completed in T ═ T0+ [465 to 1105] seconds.

Referring now to fig. 6I, there is shown a post-RCA polymerization addressing phase that typically occurs after completion of the RCA polymerization phase of fig. 6F, the amplicon extension phase of fig. 6G, and the exponential RCA amplification phase of fig. 6H, preferably 10 to 300 volts/cm in the presence of a DC electric field, preferably in the same direction as the electric field in the step of fig. 6H. The post-RCA polymerization addressing stage is particularly useful for collecting the plurality of amplicons 640 in the solution 602, the plurality of amplicons 640 being at a distance from the plurality of target molecule-specific microgel deposits 170 and recapturing the plurality of amplicons 640 at the plurality of target molecule-specific microgel deposits 170, as indicated by the plurality of arrows 660, and preferably, concentrating the plurality of amplicons 640 at one location within each microgel region 620. The duration of the post-RCA polymerization addressing phase is typically 10 seconds to 30 seconds. Preferably, the post-RCA polymerisation addressing stage is completed in T ═ T0+ [475 to 1135] seconds.

Referring now to fig. 6J, there is shown a reporting phase generally following the post RCA aggregation addressing phase. Preferably, the reporting phase occurs in the presence of a fluorescent reporter 670 complementary to the plurality of amplicons 640, 650, which is introduced into the electrophoretic array assembly 600 via a solution. The duration of the reporting phase is typically 10 to 30 seconds. Preferably, the reporting phase is completed in T ═ T0+ [485 to 1165] seconds.

After completion of the reporting phase and a subsequent washing phase (not shown), the presence of at least one nucleic acid target molecule can be detected from a plurality of pre-selected nucleic acid target molecules via conventional fluorescence detection. Thus, it should be understood that preferably the detection of at least one nucleic acid target molecule should be completed within 8 minutes to 20 minutes of the initial supply of solution 602 to the interior of the electrophoretic array assembly 600.

It should be understood that if the preparation of the solution 602 is completed within 4 minutes to 5 minutes of taking a sample, for example: by taking a blood sample from a patient, at least one nucleic acid target molecule can be detected from the sample in 12 minutes to 25 minutes.

Referring now to fig. 7A-7J, fig. 7A-7J illustrate major stages in rapid detection of the presence of at least one nucleic acid target molecule from a plurality of pre-selected nucleic acid target molecules, according to another preferred embodiment of the present invention. It should be understood that the substrate 110, the perimeter wall structure 120, and the window 130, as well as the various layers making up the electrophoretic array 160, are not shown for simplicity. Each of fig. 7A-7J is a simplified side cross-sectional view taken generally along lines 4-4 in fig. 2A.

Fig. 7A shows an electrophoretic array assembly 700 similar to that shown in fig. 2A in a dried, dehydrated, operational orientation, the electrophoretic array assembly 700 having indications of a symbolic variety of different oligonucleotide forward primers 322, a plurality of nucleic acid target molecule-specific RCA loop probes 320, and a plurality of reverse primers 324, the plurality of reverse primers 324 being associated with a corresponding plurality of different dried microgel deposits 190. Fig. 7A shows the internal dimensions of a preferred embodiment of the electrophoresis array assembly 700 in millimeters, along with the general dimensions of the plurality of immobilized dried target molecule-specific microgel deposits 190.

It should be understood that the method of fig. 7A to 7J is different from the method of fig. 4A to 4J in that, instead of the plurality of capture probes 400 being bound to the plurality of immobilized dry target molecule-specific microgel deposits 190 in the method of fig. 4A to 4J, a plurality of nucleic acid target molecule-specific RCA loop probes 320 are specifically hybridized to a plurality of forward primers 322, and a plurality of forward primers 322 are bound to the plurality of immobilized dry target molecule-specific microgel deposits 190, and a plurality of reverse primers 324 are also bound to the plurality of immobilized dry target molecule-specific microgel deposits 190 in the method of fig. 7A to 7J.

FIG. 7B shows the introduction of a solution 702 containing a plurality of nucleic acid target molecules 703, represented by an arrow 701, to fill the interior of the electrophoretic array assembly 700.

The preparation of the solution 702 is not part of the claimed invention, and the invention is carried out according to conventional techniques, such as "Nasir Ali, Rita de C a sia Pontello Rampazzo, Alexandre Dias Tavares Costa and Marco Aureliao Kriger, Current Nucleic Acid Extraction Methods and therapeutics to Point-of-Care Diagnostics, BioMed Research International, 2017, article number 93065". Preferably, solution 702 includes a low conductivity eluent, typically introduced during the preparation of the solution, that facilitates electronic addressing of nucleic acids and promotes the activity of restriction enzymes in solution 702. A preferred eluent comprises histidine and a restriction enzyme buffer. Fig. 7B shows the plurality of dried target molecule-specific microgel deposits 190 in a dehydrated state. The solution 702 is introduced into the electrophoresis array assembly 700 such that the solution 702 contacts the plurality of dried target molecule-specific microgel deposits 190, defines a time T0 and causes the plurality of dried target molecule-specific microgel deposits 190 to assume their hydrated state, as indicated by reference numeral 170 (fig. 1A, fig. 1B).

Fig. 7C shows a plurality of immobilized target molecule-specific microgel deposits 170 in a monohydrate state as a result of introducing a solution 702 containing a plurality of nucleic acid target molecules 703 and filling the interior of the electrophoretic array assembly 700. Fig. 7C shows the plurality of target molecule-specific electrically isolated microgel deposits 170 hydrated, the plurality of microgel deposits 170 having a typical maximum height of 1.25 millimeters. Preferably, the hydration of the plurality of target molecule-specific microgel deposits 170 to the hydration state shown in fig. 7C takes about 10 seconds, and is preferably completed at time T ═ T0+10 seconds.

Preferably, FIG. 7D shows the electrophoretic addressing of a plurality of nucleic acid target molecules 703 to the plurality of hydrated, immobilized, mutually electrically isolated target molecule-specific microgel deposits 170 in the presence of a DC electric field of 10 volts to 300 volts/cm. Portions of the plurality of electric field lines are represented by a plurality of reference numerals 710 and the direction of the electric field is represented by a plurality of arrows 712. It should be appreciated that the plurality of electric field lines define a plurality of three-dimensional, fixed, spaced apart and electrically isolated microgel zones 720, each microgel zone 720 surrounding a different target molecule-specific microgel deposit 170.

It should be understood that addressing and the various steps described below with reference to fig. 7E-7J occur not only on the surface of the plurality of microgel deposits 170, but also within the volume of the plurality of microgel deposits 170.

As shown in fig. 7D, application of the DC electric field to the plurality of three-dimensional, immobilized, spaced apart and electrically isolated target molecule-specific microgel regions 720 causes rapid transport of a plurality of nucleic acid target molecules 703 to the target molecule-specific microgel deposits 170, thereby promoting specific hybridization between the plurality of nucleic acid target molecules 703 and the plurality of nucleic acid target molecule-specific RCA loop probes 320, wherein the plurality of RCA loop probes 320 are bound to the plurality of forward primers 322, and the plurality of forward primers 322 are bound to the hydrated plurality of target molecule-specific electrically isolated microgel deposits 170.

The duration of the phase shown in fig. 7D is between 30 seconds and 120 seconds. Preferably, the phase shown in fig. 7D is completed in a time T ═ T0+ [40 to 130] seconds.

It should be understood that prior to the various stages described below with reference to fig. 7E-7J, an optional removal stage (not shown) may be added after the addressing stage shown in fig. 7D, preferably 10 to 300 volts/cm in the opposite direction to the electric field in the step of fig. 7D. Preferably, the removal stage is for removing a plurality of non-specific target molecules hybridised to the plurality of RCA loop probes 320.

Referring now to FIG. 7E, there is shown a connect phase generally following the address phase shown in FIG. 7D, preferably in the presence of a DC electric field of 10 volts to 300 volts/cm in the same direction as the electric field in the step of FIG. 7D. Preferably, the ligation phase occurs in the presence of a ligase 728 (e.g., T-4 ligase), which ligase 728 is introduced into the electrophoretic array assembly 700 via a solution. The duration of the connection phase is typically 120 to 240 seconds. Preferably, the connection phase is completed in T ═ T0+ [160 to 370] seconds.

Reference is now made to fig. 7F, which illustrates a RCA polymerisation phase generally following the ligation phase shown in fig. 7E, and preferably occurring in the presence of a DC electric field, preferably 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 7E. Preferably, the RCA polymerization stage occurs in the presence of a Bst polymerase 729 and dntps (not shown) introduced into the electrophoretic array assembly 700 through a solution, and the forward primer 322 and reverse primer 324 are bound to the target molecule-specific microgel deposit 170, preferably at a temperature of 65 ℃. One result of the RCA polymerization stage is the generation of multiple long RCA amplicons 740 (fig. 7G). As shown in fig. 7F, after polymerase 729 binds to the plurality of nucleic acid target molecule-specific RCA loop probes 320, the plurality of nucleic acid target molecules 703 are displaced from the plurality of nucleic acid target molecule-specific RCA loop probes 320, as indicated by the plurality of arrows 742. The duration of the RCA polymerisation stage is typically 300 to 720 seconds. Preferably, the RCA polymerisation stage is completed in T ═ T0+ [460 to 1090] seconds.

Referring now to fig. 7G, there is shown an amplicon elongation and compression phase that typically occurs during the RCA polymerization phase of fig. 7F, preferably 10 to 300 volts/cm in the same and opposite directions as the electric field in the step of fig. 7F, as indicated by an arrow 744. Elongation of the amplicon 740 occurs in the direction indicated by an arrow 748. Compression generally occurs in a direction opposite to that shown by arrow 748. Amplicon elongation and compression enhances hybridization of the plurality of reverse primers 324, the plurality of reverse primers 324 binding to the plurality of target molecule-specific microgel deposits 170, amplicons 740. Preferably, the amplicon elongation and compression stages occur in the presence of a Bst polymerase 729, dntps (not shown) and at a temperature of 65 ℃. The duration of the amplicon extension phase is typically 5 to 15 seconds. Preferably, the RCA polymerization stage is completed in T ═ T0+ [ 465-.

It should be understood that the stages shown in fig. 7F and 7G may be repeated intermittently a plurality of times, each stage of the plurality of stages shown in fig. 7F being shorter in duration than described above and separated by a stage shown in fig. 7G.

Referring now to fig. 7H, there is shown an exponential RCA amplification phase that occurs generally during the RCA polymerization phase of fig. 7F and the amplicon compression extension phase of fig. 7G, and preferably in the presence of a DC electric field, preferably generally 10 to 300 volts/cm in a direction opposite the electric field in the step of fig. 7G, but preferably including short periods of reversal of polarity of the electric field. At this stage, a plurality of reverse primers 324 are used to generate a plurality of additional amplicons 750.

Preferably, the exponential RCA amplification stage one occurs in the presence of Bst polymerase 729, a plurality of dntps (not shown), and reverse primer 324 at a temperature of 65 ℃. The duration of the exponential RCA amplification phase occurring during the RCA polymerization phase of fig. 7F and the amplicon extension phase of fig. 7G is typically 5 to 15 seconds. Preferably, the RCA polymerization stage of fig. 7F, the amplicon extension stage of fig. 7G, and the exponential RCA amplification stage of fig. 7H are completed in T ═ T0+ [465 to 1105] seconds.

Referring now to fig. 7I, there is shown a post-RCA amplicon concentration phase that occurs generally after completion of the RCA polymerization phase of fig. 7F, the amplicon elongation and compression phase of fig. 7G, and the exponential RCA amplification phase of fig. 7H, and preferably in the presence of a DC electric field, preferably generally 10 to 300 volts/cm in the same direction as the electric field in the step of fig. 7H. The amplicon concentration stage is particularly suitable for concentrating a plurality of amplicons 740 and 750 at a location within each microgel region 720, as indicated by a plurality of arrows 760. The amplicon concentration phase typically has a duration of 10 seconds to 30 seconds. Preferably, the post-RCA polymerisation addressing stage is completed in T ═ T0+ [475 to 1135] seconds.

Referring now to fig. 7J, a reporting phase is shown generally following the post-RCA polymerization addressing. Preferably, the reporting phase occurs in the presence of a fluorescent reporter 770 that is complementary to the plurality of amplicons 740, 750, which are introduced into the electrophoretic array assembly 700 through a solution. The duration of the reporting phase is typically 10 to 30 seconds. Preferably, the reporting phase is completed in T ═ T0+ [485 to 1165] seconds.

After completion of the reporting phase and a subsequent washing phase (not shown), the presence of at least one nucleic acid target molecule can be detected from a plurality of pre-selected nucleic acid target molecules via conventional fluorescence detection. Thus, it should be understood that preferably the detection of at least one nucleic acid target molecule should be completed within 8 minutes to 20 minutes of the initial supply of solution 702 to the interior of the electrophoretic array assembly 100.

It will be appreciated that if the preparation of the solution 701 is completed within 4 minutes to 5 minutes of taking a sample, for example: by taking a blood sample from a patient, at least one nucleic acid target molecule can be detected from the sample in 12 minutes to 25 minutes.

Several examples

Example 1: the method of FIGS. 7A to 7J was used to detect the causative agent of meningitis and a synthetic DNA target molecule representing Neisseria meningitidis (Neisseria meningitidis) was used.

An electrophoresis array assembly similar to the electrophoresis array assembly 700 (fig. 7A) is provided, comprising 100 immobilized dried target molecule-specific microgel deposits 190, the immobilized dried target molecule-specific microgel deposits 190 having a base diameter of 0.45 mm and a height of about 0.2 mm to 0.3 mm. The 48 immobilized dried target molecule-specific microgel deposits 190 are spotted as a plurality of RCA loop probes 320, which plurality of RCA loop probes 320 are pre-hybridized with a plurality of forward primers 322, and a plurality of reverse primers 324 specific for 9 different pathogen DNA targets, each of which is associated with the detection of meningitis, including especially diplococcus meningitidis, are used. Thus, the 48 point-like immobilized dried target molecule-specific microgel deposits 190 are target molecule-specific, as shown below:

1, deposition: specific for meningococcus

And (3) deposition 2: specific for meningococcus

And (3) deposition: specific for meningococcus

And 4, deposition: specific for Escherichia coli

And (5) deposition: specific for Escherichia coli

And 6, deposition: specific for Escherichia coli

And 7, deposition: specific for meningococcus

And (4) deposition of 8: specific for meningococcus

And (4) deposition 9: specific for meningococcus

And (4) deposition 10: specific for enteroviruses

Deposit 11: specific for enteroviruses

Deposition 12: specific for meningococcus

And (3) deposition of 13: specific for meningococcus

Deposit 14: specific for meningococcus

15, deposition: group B streptococcus

And (3) deposition 16: group B streptococcus

Deposit 17: group B streptococcus

Deposition 18: specific for meningococcus

Deposit 19: specific for meningococcus

And (4) deposition 20: specific for meningococcus

Deposit 21: specific for Haemophilus influenzae

Deposit 22: specific for Haemophilus influenzae

Deposit 23: specific for Haemophilus influenzae

Deposit 24: specific for meningococcus

Deposit 25: specific for meningococcus

Deposit 26: specific for meningococcus

Deposit 27: specific for human herpesvirus

Deposit 28: specific for human herpesvirus

Deposition 29: specific for human herpesvirus

30, deposition: specific for human herpesvirus

Deposition 31: specific for meningococcus

Deposit 32: specific for meningococcus

Deposit 33: specific for meningococcus

Deposit 34: specific for human paraenterovirus

Deposit 35: specific for human paraenterovirus

Deposit 36: specific for human paraenterovirus

Deposit 37: specific for meningococcus

Deposition 38: specific for meningococcus

Deposit 39: specific for meningococcus

And (4) deposition 40: specific for Listeria

Deposition 41: specific for Listeria

Deposit 42: specific for Listeria

Deposit 43: specific for meningococcus

Deposit 44: specific for meningococcus

Deposit 45: specific for meningococcus

Deposition 46: is specific to varicella zoster virus

And (4) deposition 47: is specific to varicella zoster virus

And (3) deposition 48: is specific to varicella zoster virus

At a time point defined as T0, a solution 702 containing a plurality of nucleic acid target molecules 703(100 nanomolar/liter concentration) representative of neisseria meningitidis is provided to the interior volume of the electrophoresis array. The solution 702 also includes a low conductivity buffer that supports rapid DNA transport and hybridization with the RCA probes deposited on the microgels.

After a duration of one 10 seconds, the solution 702 is supplied so that the plurality of dried target molecule-specific microgel deposits 190 assume their hydrated state, indicated by reference numeral 170 (fig. 7B to 7C).

At time T-T0 +10 seconds, a constant current of 1.6 milliamps is applied to the working and counter electrodes, respectively, at points 260 and 250, producing a voltage of 4.5 volts, thereby applying an electric field of 12.5 volts/cm across the electrophoretic array and producing electrophoretic addressing (fig. 7D). The duration of the electrophoretic addressing is 40 seconds.

At time T ═ T0+50 seconds, a ligation reaction solution containing ligation enzyme T4 ligase (Blunt T/a, from the biological laboratory in new england) was supplied to the internal volume of the electrophoresis array, replacement solution 702, for approximately 180 seconds (fig. 7E).

At time T-T0 +230 seconds, a polymerase solution containing Bst polymerase 729 and dntps (from the biological laboratory in new england) was supplied to the internal volume of the electrophoresis array, displacing the ligation reaction solution for approximately 720 seconds (fig. 7F).

At time T-T0 +950 seconds, a constant current of 1.6 milliamps was applied to the working and counter electrode contacts 260 and 250, respectively, creating a voltage of 4.5 volts, thereby applying an electric field of 12.5 volts/cm across the electrophoretic array and providing recapture of RCA amplicons from the polymerase solution. The duration of this step is about 20 seconds (fig. 7I).

A red reporter solution (Alexa 647 from Integrated Device Technology, inc., San Jose, CA) containing fluorescently labeled oligonucleotides was supplied to the interior volume of the electrophoresis array at time T0+970 seconds, replacing the polymerase solution for approximately 30 seconds (fig. 7J). After washing away the red reporter solution, a fluorescent image of the electrophoretic array assembly 700 is taken through the window 130 and the presence of nucleic acid target molecules 703 representing neisseria meningitidis is detected in the following multiple fixed dried target molecule-specific microgel deposits: 1.2, 3, 7, 8, 9, 13, 14, 15, 19, 20, 21, 25, 26, 27, 31, 32, 33, 37, 38, 39, 43, 44 and 45. The presence of the nucleic acid target molecule 703 representing neisseria meningitidis was not detected in the following plurality of immobilized dried target molecule-specific microgel deposits 170: 4.5, 6, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35, 36, 40, 41, 42, 46, 47, and 48.

The results of the test are summarized in FIG. 8. It should be noted that the average ratio of the intensity of the fluorescent signals obtained from the plurality of deposits 1, 2, 3, 7, 8, 9, 13, 14, 15, 19, 20, 21, 25, 26, 27, 31, 32, 33, 37, 38, 39, 43, 44, and 45 to the intensity of the fluorescent signals from the plurality of deposits 4, 5, 6, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35, 36, 40, 41, 42, 46, 47, and 48 is about 8.5.

Example 2:

the method of fig. 7A-7J was used to detect meningitis pathogens and a genomic DNA target molecule pathogen extracted from neisseria meningitidis was used to incorporate into clinical samples of cerebrospinal fluid.

An electrophoresis array assembly similar to the electrophoresis array assembly 700 (fig. 7A) is provided, which comprises 100 immobilized dried target molecule-specific microgel deposits 190, the plurality of immobilized dried target molecule-specific microgel deposits 190 having a base diameter of 0.45 mm and a height of about 0.2 mm to 0.3 mm. The 21 immobilized dried target molecule-specific microgel deposits 190 are spotted as a plurality of RCA loop probes 320, which plurality of RCA loop probes 320 are pre-hybridized with a plurality of forward primers 322, and reverse primers 324 specific for 9 different pathogen DNA targets are used, each target being associated with the detection of meningitis, including especially diplococcus meningitidis. Thus, the 21 spotted immobilized dried target molecule-specific microgel deposits 190 are target molecule-specific, as shown below:

1, deposition: specific for Escherichia coli

And (3) deposition 2: specific for Escherichia coli

And (3) deposition: specific for meningococcus

And 4, deposition: specific for meningococcus

And (5) deposition: specific for meningococcus

And 6, deposition: specific for enteroviruses

And 7, deposition: specific for enteroviruses

And (4) deposition of 8: specific for meningococcus

And (4) deposition 9: specific for meningococcus

And (4) deposition 10: specific for meningococcus

Deposit 11: group B streptococcus

Deposition 12: group B streptococcus

And (3) deposition of 13: specific for Haemophilus influenzae

Deposit 14: specific for Haemophilus influenzae

15, deposition: specific for meningococcus

And (3) deposition 16: specific for meningococcus

Deposit 17: specific for meningococcus

Deposition 18: specific for Listeria

Deposit 19: specific for Listeria

And (4) deposition 20: is specific to varicella zoster virus

Deposit 21: is specific to varicella zoster virus

A clinical sample of cerebrospinal fluid (CSF) was spiked with meningococcal pathogens and genomic DNA was extracted using a common magnetic bead-based DNA extraction method. The input concentration of DNA targets in cerebrospinal fluid was determined by a reference real-time PCR method that yielded a pathogen concentration of neisseria meningitidis in a clinical sample of 720 DNA-replicating cerebrospinal fluid per microliter of CSF.

At a point in time defined as T0, a solution 702 is supplied to the internal volume of the electrophoretic array, the solution 702 being prepared from the spiked clinical sample. The solution 702 also includes a low conductivity buffer that supports rapid DNA transport and hybridization with the RCA probes deposited on the microgels.

After a duration of one 10 seconds, the solution 702 is supplied so that the plurality of dried target molecule-specific microgel deposits 190 assume their hydrated state, indicated by reference numeral 170 (fig. 7B to 7C).

At time T-T0 +10 seconds, a constant current of 1.6 milliamps is applied to the working and counter electrode contacts 260 and 250, respectively, resulting in a voltage of 4.5 volts, thereby applying an electric field of 12.5 volts/cm across the electrophoretic array and resulting electrophoretic addressing (fig. 7D). The duration of the electrophoretic addressing is 40 seconds.

At time T-T0 +50 seconds, an electric field of reversed polarity of 1.6 milliamperes was applied across the working and counter electrode contacts 260 and 250, respectively, resulting in a voltage of 4.5 volts, thereby applying an electric field of 12.5 volts/cm across the electrophoretic array and enhancing removal of non-specifically bound DNA targets. The duration of the electrophoretic addressing was 10 seconds (fig. 7G).

At time T ═ T0+60 seconds, a ligation reaction solution containing ligation enzyme T4 ligase (Blunt T/a, from the biological laboratory in new england) was supplied to the internal volume of the electrophoresis array, replacement solution 702, for a duration of approximately 180 seconds (fig. 7E).

At time T ═ T0+240 seconds, a polymerase solution containing Bst polymerase 729 and dntps (from the biological laboratory in new england) was supplied to the internal volume of the electrophoresis array, displacing the ligation reaction solution, for a duration of approximately 720 seconds (fig. 7F).

At time T-T0 +960 seconds, a constant current of 1.6 milliamps was applied to the working and counter electrode contacts 260 and 250, respectively, resulting in a voltage of 4.5 volts, thereby applying an electric field of 12.5 volts/cm across the electrophoretic array and providing recapture of RCA amplicons from the polymerase solution. The duration of this step is about 20 seconds (fig. 7I).

A red reporter solution (Alexa 647 from Integrated Device Technology, inc., San Jose, CA) containing fluorescently labeled oligonucleotides was supplied to the interior volume of the electrophoresis array at time T0+980 seconds, replacing the polymerase solution for approximately 30 seconds (fig. 7J). After washing away the red reporter solution, a fluorescent image of the electrophoretic array assembly 700 is taken through the window 130 and the presence of nucleic acid target molecules 703 representing neisseria meningitidis is detected in the following multiple fixed dried target molecule-specific microgel deposits: 3. 4, 5,8, 9, 10, 15, 16 and 17. The presence of the nucleic acid target molecule 703 representing neisseria meningitidis was not detected in the following plurality of immobilized dried target molecule-specific microgel deposits 170: 1.2, 6, 7, 11, 12, 13, 14, 18, 19, 20 and 21.

The results of the test are summarized in FIG. 9. It should be noted that the average ratio of the intensity of the fluorescent signals obtained from the plurality of deposits 3, 4, 5,8, 9, 10, 15, 16 and 17 to the intensity of the fluorescent signals from deposits 1, 2, 6, 7, 11, 12, 13, 14, 18, 19, 20 and 21 is about 4.3.

It will be understood by those skilled in the art that the present invention is not limited to what has been described and illustrated herein, but also includes combinations and sub-combinations of the various features described herein, as well as various modifications thereof which are not in the prior art.