阅读说明：本技术 一种3D DNA行走机器耦合催化发夹自组装的microRNA生物传感器 (3D DNA walking machine coupling catalysis hairpin self-assembly microRNA biosensor ) 是由 谢国明 杨廷燕 方杰 于 2019-05-20 设计创作，主要内容包括：在本专利中,我们提出了一种新的、熵驱动的三维DNA步行机器,它结合了催化发夹组装反应(CHA)成功的用于检测microRNA。此步行机器使用了链霉亲合素包裹的聚苯乙烯微球作为3-D轨道基质,以保证良好的重复性。其制备方法是将miRNA-21作为一个靶目标,通过熵驱动链置换反应不断地在DNA功能化的聚苯乙烯微球轨道上行走,导致聚苯乙烯微球上DNA三核酸复合底物的分解,从而实现miRNA 21的循环。此外,从DNA三核酸复合底物中释放大量辅助链可以催化CHA反应并伴随着获得荧光信号的增加。这种复合生物传感器的线性范围在50pM-20nM之间,检测限低至41pM。此外,在重复性试验(1.05％至4.22％)和回收率试验(99.5％至104.8％)中均获得了满意的结果。结果表明,该方法在疾病诊断方面具有潜在的应用价值。(In this patent, we propose a new, entropy-driven, three-dimensional DNA walking machine that incorporates the catalytic hairpin assembly reaction (CHA) for the successful detection of micrornas. The walking machine used streptavidin-coated polystyrene microspheres as the 3-D orbital matrix to ensure good reproducibility. The preparation method comprises the steps of taking miRNA-21 as a target, continuously walking on a polystyrene microsphere track with DNA functionalization through entropy-driven strand displacement reaction, and causing decomposition of a DNA trinucleotide composite substrate on the polystyrene microsphere, so that circulation of miRNA21 is realized. In addition, the release of a large number of helper strands from the DNA trinucleotide complex substrate may catalyze the CHA reaction with a concomitant increase in the fluorescent signal obtained. The linear range of the composite biosensor is between 50pM and 20nM, and the detection limit is as low as 41 pM. In addition, satisfactory results were obtained in both the reproducibility test (1.05% to 4.22%) and the recovery test (99.5% to 104.8%). The result shows that the method has potential application value in disease diagnosis.)

1. A simple entropy-driven 3-D DNA walking machine coupling catalysis hairpin self-assembly reaction biosensor for microRNA detection is provided, and the preparation method comprises the following steps:

(1) preparation of 3-D DNA Walking machine

The purified three nucleic acid chains LS, AS, S and TEMg buffer are annealed and then refolded to prepare the LS/AS/S trinucleotide complex (C3). The ratio of LS to AS to S is 1: 1.5. The annealing process was carried out by heating the solution to 95 ℃ for 5min in a PCR instrument and then slowly lowering the temperature to 25 ℃ at a rate of 0.1 ℃ per second. Then 50. mu.L of streptavidin-modified polystyrene microspheres (PS) were centrifuged at 12000 rpm for 5 minutes, washed 2 times with binding buffer and centrifuged to discard the supernatant. Resuspend with 50. mu.L binding buffer and add 5. mu.L. mu. M C3. After incubation in an incubator at 37 ℃ for 15 minutes, the supernatant was discarded by washing 3 times with TEMg buffer and centrifuging at 12000 rpm for 5 minutes. Finally, resuspend with 50. mu.L TEMg buffer, and finally prepare the DNA functionalized 3-D DNA walking machine (C3/PS).

(2) Preparation of Signal probes

The hairpin probe (H1) and hairpin probe (H2) were annealed and refolded. The solution was heated to 95 ℃ for 5min in a PCR instrument and then slowly lowered to 25 ℃ at a rate of 0.1 ℃ per second. A FAM-labeled signaling probe RepF and a BHQ 1-labeled signaling probe RepQ were hybridized at a ratio of 1: 2 in 1 XTNaK buffer.

(3) Detection of MicroRNA

Mu. L C3/PS complex, 2. mu.L of 10. mu.M fuel nucleic acid (FS), 33. mu.L of cutscart buffer, and 10. mu.L of various concentrations of target were incubated at 37 ℃ for 60 minutes in an incubator. Then 40. mu.L of the supernatant was centrifuged and added to a solution containing 5. mu.L of 1. mu.MH 1, 2. mu.L of 10. mu. M H2, 10. mu.L of 50nM RepF: RepQ complex and 23. mu.L of 1 XTNaK buffer were reacted for 60 minutes at room temperature in the absence of light. And finally, measuring the fluorescence signal by using a fluorescence spectrophotometer.

2. The biosensor of claim 1, wherein the ratio of LS to AS to S in step (1) is 1: 1.5.

3. The entropy-driven 3-D DNA walking machine-coupled biosensor that catalyzes hairpin self-assembly reaction according to claim 1, which is prepared by a method in which the ratio of RepF to RepQ in step (2) is 1: 2.

4. The biosensor of claim 1, wherein the entropy-driven 3-D DNA walking machine-coupled catalyzed hairpin self-assembly reaction is prepared by incubating at 37 ℃ for 60min in step (3).

5. The entropy-driven 3-D DNA walking machine-coupled catalysis hairpin self-assembly reaction biosensor as claimed in claim 1, wherein the preparation method comprises the step (3) of carrying out the reaction at room temperature for 60 minutes in a dark place.

Technical Field

The invention relates to a biosensor, in particular to a biosensor for microRNA detection, which is simple and convenient and is driven by entropy and is used for 3-D DNA walking machine coupling catalysis hairpin self-assembly reaction

Background

The DNA machine is a molecular machine constructed from DNA that can be logically designed due to the versatility and predictability of the Watson Crick base-pairing rules. Various DNA machines are constructed and synthesized by highly programmable methods, including DNA tweezers, DNA motors, DNA walkers, and DNA robots. Among them, DNA walkers can perform precise control in microscale or nanoscale programmed oligonucleotide orbits, and have important application potential in biosensors, biometrics, and drug delivery. Previous studies have reported movement of DNA walkers along one-dimensional (1D) tracks, two-dimensional (2D) origami, or three-dimensional (3D) tracks. Among them, 3D DNA walkers have more powerful signal amplification capabilities due to all their reactions confined to micron-or nanoscale materials, increasing the local concentration of reactants resulting in more stable signal output. Therefore, the use of 3D DNA walkers has attracted a great deal of attention. For example, Li and coworkers have designed an enzyme-driven DNA walker sensor with a three-dimensional trajectory that enables the detection of specific nucleic acids; fan et al designed a random DNA walker driven by exonuclease III for ultrasensitive bioanalysis and cascade signal amplification. Liu and his colleagues described a three-dimensional DNA walker for the detection of 8-hydroxy-2' -deoxyguanosine, which was three orders of magnitude lower than previously reported methods. Although these methods have high sensitivity, they are enzyme-dependent, susceptible to complex conditions (e.g., buffer and temperature), costly to detect, and not amenable to long-term storage. Therefore, the study of non-enzymatic three-dimensional DNA walkers has attracted considerable attention.

Recently, Ye and his colleagues reported a three-dimensional DNA walker walking on gold nanoparticles (AuNPs) promoted by entropy-driven reaction (EDR). In contrast to enzyme-dependent signal amplification systems, entropy-driven reactions are a catalytic signal amplification strategy that does not require enzymes or precise temperature cycling. In the case of an increase in entropy, multiple functions are exerted in the catalytic circuit by a toehold-mediated strand displacement reaction using a string of single nucleic acid strands. Due to the unique and remarkable driving force, the total base number of the system is kept unchanged in the amplification process, so that the chance of circuit leakage is reduced, and a more reliable platform is provided for accurate detection. With the help of EDR, the above three-dimensional DNA walker has a higher sensitivity in imaging of mirnas. However, AuNPs have inevitable limitations as three-dimensional orbital matrices. On the one hand, the interaction between DNA and AuNPs is usually via gold-sulfur bonds. This linkage is not strong enough to avoid non-specific DNA adsorption, which severely hinders DNA hybridization and reduces colloidal stability. On the other hand, it requires immobilization of affinity ligand and trinucleotide complexes on the AuNPs surface and corresponding optimization of their ratio, resulting in poor reproducibility of DNA and AuNPs conjugate products.

The ratio of (A) and (B) is optimized correspondingly, so that the repeatability of the DNA and AuNPs conjugate product is poor.

One possible solution to the above problem is to find other substrates for immobilizing DNA. The monodisperse polystyrene microsphere has large surface area and strong surface reaction capability, and is widely applied to DNA bar code detection. More importantly, the commercial streptavidin modified polystyrene microsphere can be easily functionalized through avidin-biotin reaction, and has good repeatability. Streptavidin binds much more strongly to biotin than the Au-S bond. Therefore, the streptavidin-coated polystyrene microspheres can be used as a potential substrate for constructing three-dimensional DNA walkers. Meanwhile, the use of isothermal amplification technology in the construction of DNA machinery to achieve sensitive detection of biomolecules has attracted increasing interest. Catalytic hairpin self-assembly (CHA) is an economical and efficient enzyme-free cascade amplification strategy. The advantages of entropy-driven based three-dimensional DNA walkers and CHAs have therefore led to our interest in the field of nucleic acid detection.

In the current work, a new biosensor based on coupling of entropy-driven three-dimensional DNA walkers and CHA catalytic hairpin self-assembly reaction is provided, and sensitive detection on picomolar miRNA is realized. We used miRNA21 as a model target, which is closely related to many diseases including hematologic cancers. In the system, miRNA21 is used AS a catalyst and a random DNA walker, and continuously moves on the surface of a polystyrene microsphere, so that a trinucleotide complex fixed on the microsphere is decomposed, a large amount of auxiliary nucleic Acid (AS) is released, and the circulation of miRNA21 is realized. At the same time, AS may catalyze the CHA reaction to produce a high signal output. Due to the introduction of entropy-driven reaction, it has significant selectivity in distinguishing from other homologous miRNAs, and the signals of several miRNAs except miRNA-200b account for less than 5% of the signal of miR-121. The composite biosensor can work in the concentration range of 50pM to 20nM, and the detection limit is as low as 41 pM. In addition, satisfactory results were obtained in both the repeatability tests (from 1.05% to 4.22%) and the recovery tests (from 99.5% to 104.8%).

Disclosure of Invention

The invention aims to develop a biosensor for simply, specifically and sensitively detecting RNA.

The specific technical scheme is as follows:

a simple entropy-driven 3-D DNA walking machine coupling catalysis hairpin self-assembly reaction biosensor for microRNA detection is provided, and the preparation method comprises the following steps:

(1) preparation of 3-D DNA Walking machine

The purified three nucleic acid chains LS, AS, S and TEMg buffer are annealed and then refolded to prepare the LS/AS/S trinucleotide complex (C3). The ratio of LS to AS to S is 1: 1.5. The annealing process was carried out by heating the solution to 95 ℃ for 5min in a PCR instrument and then slowly lowering the temperature to 25 ℃ at a rate of 0.1 ℃ per second. Then 50. mu.L of streptavidin-modified polystyrene microspheres (PS) were centrifuged at 12000 rpm for 5 minutes, washed 2 times with binding buffer and centrifuged to discard the supernatant. Resuspend with 50. mu.L binding buffer and add 5. mu.L. mu. M C3. After incubation in an incubator at 37 ℃ for 15 minutes, the supernatant was discarded by washing 3 times with TEMg buffer and centrifuging at 12000 rpm for 5 minutes. Finally, resuspend with 50. mu.L TEMg buffer, and finally prepare the DNA functionalized 3-D DNA walking machine (C3/PS).

(2) Preparation of Signal Probe

The hairpin probe (H1) and hairpin probe (H2) were annealed and refolded. The solution was heated to 95 ℃ for 5min in a PCR instrument and then slowly lowered to 25 ℃ at a rate of 0.1 ℃ per second. A FAM-labeled signaling probe RepF and a BHQ 1-labeled signaling probe RepQ were hybridized at a ratio of 1: 2 in 1 XTNaK buffer.

(3) Detection of MicroRNA

Mu. L C3/PS complex, 2. mu.L, 10. mu.M fuel nucleic acid (FS), 33. mu.L of cutsmart buffer, and 10. mu.L of various concentrations of target were incubated for 60 minutes at 37 ℃ in an incubator. Then 40. mu.L of the supernatant was centrifuged and added to a solution containing 5. mu.L of 1. mu. M H1, 2. mu.L of 10. mu. M H2, 10. mu.L of 50nM RepF: RepQ complex and 23. mu.L of 1 XTNaK buffer were reacted for 60 minutes at room temperature in the absence of light. And finally, measuring the fluorescence signal by using a fluorescence spectrophotometer.

The ratio of LS to AS to S in the step (1) is 1: 1.5.

The ratio of RepF to RepQ in the step (2) is 1: 2.

The entropy driving in the step (3) is to incubate in a constant temperature oven at 37 ℃ for 60 minutes

The self-assembly reaction of the catalysis hairpin in the step (3) is carried out for 60 minutes in a dark place at room temperature

The invention establishes a 3D DNA walking machine taking polystyrene microspheres as tracks and couples with a catalytic hairpin self-assembly reaction to sensitively detect a biosensor of miRNA. The detection principle is that the connecting nucleic acid (LS), the auxiliary nucleic Acid (AS) and the matrix nucleic acid (S) can form a trinucleotide complex (C3) by the base complementary pairing principle, biotin is modified at the 5' end of the connecting nucleic acid (LS), and the trinucleotide complex can be fixed on a polystyrene microsphere modified with streptavidin through a streptavidin reaction. In the presence of a target miRNA21 (T), AS is replaced by a tohelld-mediated strand displacement reaction, a new tohelld is exposed, and the target miRNA21 can be replaced by the new tohelld in the presence of a fuel nucleic acid (F) to form a cycle and generate a waste double-stranded F/S. In addition, the displaced AS can be used AS a catalyst chain to catalyze the subsequent self-assembly reaction of the catalytic hairpin, the hairpin structure H1 is opened, and then the hairpin structure H2 is opened, an H1/H2 compound is formed, and the cycle of AS is formed to open new H1 and H2. One nucleic acid chain labeled with FAM fluorescent group and one nucleic acid chain labeled with BHQ1 quenching group form a double chain through base complementary pairing, and form a signal probe. The nucleic acid chain marked with FAM fluorescent group can be replaced by toheld of the H1 and H2 complex, so that quenched fluorescence is recovered, and a fluorescence signal of the fluorescence is detected by a fluorescence spectrophotometer.

The shape uniformity of the polystyrene microspheres plays an important role in the repeatability of the 3D DNA walking machine, and the polystyrene microspheres are morphologically characterized by adopting a scanning electron microscope. As shown in fig. 1, a and b show the scanned images of polystyrene microspheres at low and high magnification, respectively. It can be seen that the polystyrene microspheres have a high degree of uniformity and good dispersibility. The average size is about 800 nm. The high-quality polystyrene microspheres ensure the good performance of the 3D DNA walking machine. Meanwhile, the patent proves the feasibility of the sensing strategy. First we separately demonstrate the feasibility of entropy-driven strand displacement reactions and catalyzed hairpin self-assembly reactions. FAM was labeled at the 5 'end of LS and BHQ1 was labeled at the 3' end of S, respectively. When the trinuclear complex is formed, FAM fluorescence is quenched by BHQ 1. Curve b in FIG. 2 shows that the trinuclear complex is dissociated and the quenched fluorescence is recovered when T and F are present. Curve a represents the background fluorescence signal without T. As shown in FIG. 3, the feasibility of the EDR and CHA reactions was verified on a 3.5% agarose gel, respectively. The slow migration of C3 in Lane 2 clearly indicates the formation of a trinucleotide complex. Lane 5 appeared with a new band F/S compared to Lane 3 with only C3 and F, and the band at C3 disappeared significantly, indicating that the trinucleotide complex was consumed by T. At the same time, bands of AS and H1/H2 complex appeared in lane 9, probably due to the fact that AS catalyzes the CHA reaction. In addition, other bands are used as markers or references. The feasibility of the EDR and CHA reactions was initially verified by the results of 3.5% agarose gel electrophoresis.

Subsequently, we demonstrated the feasibility of entropy-driven 3D DNA walking machine-coupled catalyzed hairpin self-assembly reactions. As shown in FIG. 4, in the absence of the entropy-driven process, the background fluorescence signal of CHA (curve a) is negligible. Furthermore, in the absence of T, there was a slight increase in background fluorescence signal when entropy-driven is combined with catalytic hairpin self-assembly (curve b). Compared to this background signal, the fluorescence signal at 520nm increased significantly in the presence of 10nm T (curve c), thus demonstrating that a binding strategy that catalyzes the entropy-driven and hairpin self-assembly is feasible. It is noteworthy that the signal-to-noise ratio of the entropy-driven process alone (fig. 2) is indeed lower than that of the entropy-driven combined biosensor that catalyzes the hairpin self-assembly reaction (fig. 4), with better signal amplification efficiency and higher efficiency. The fluorescence kinetics of the reaction system are shown in FIG. 5.

The detection conditions are important factors affecting the analytical performance of the biosensor. In this system, the number of bases of toehold 1 affects the occurrence of strand displacement reaction. We optimized the number of toehold of 7nt-10nt respectively, and these results (FIG. 6) showed that the fluorescence intensity of the walking sensor increased with the increase of the base length of toehold 1. However, when the number of bases exceeds 9nt, the signal decreases rapidly. Therefore, we chose 9nt as the optimal number of toehold for the next experiment. The concentration of F influences the feasibility of entropy driving to some extent, and we recorded the fluorescence intensities of 50nM, 100nM, 150nM, 200nM, 300 nMF. As shown in fig. 7, 200nM is the most suitable concentration, since an excess of F may result in a higher background signal. The sensor was then optimized at 25 ℃ and 37 ℃ respectively. As shown in fig. 8, the signal-to-noise ratio at 25 ℃ was higher than 37 ℃, ultimately optimizing the catalytic hairpin self-assembly (fig. 9) and entropy-driven (fig. 10) reaction times. Within the time range of 0-60 min, the signal-to-noise ratio is continuously increased along with the time, and then the signal-to-noise ratio is gradually reduced along with the increase of the time. The reason for this may be that the background signal increases more rapidly than the target signal when the time exceeds 60 minutes. Therefore, 60 minutes is considered as the optimum time.

To explore the sensitivity of this integrated fluorescent biosensor, we studied this strategy with a range of different concentrations of miRNA under optimal conditions. As shown in fig. 11, with increasing miRNA concentration, a gradual increase in fluorescence signal at 520nm was seen. In addition, the fluorescence signal varied linearly with the logarithm of the miRNA concentration in the range of 50pM to 20nM (FIG. 12), with a relatively good linear relationship. The linear regression equation was F126.651 +66.460log [ C ] (nM) (C is miRNA concentration, F is fluorescence intensity), and the correlation coefficient (R2) was 0.997. By calculation, the lowest limit of detection (LOD) was 41 pM. Comparison with other methods as shown in fig. 13 illustrates the advantage of this strategy over some existing methods.

Specificity is well known to be one of the requirements for evaluating the success or otherwise of a biosensing strategy. Therefore, we investigated the specificity of this strategy in the presence of four homologous miRNAs (including let-7d, miR-141, miR-15a, miR-200b) under identical conditions. As shown in FIG. 14, Δ F520nm shows a strong signal in the presence of 10nm miR-21. We defined Δ F520nm as Δ F520nm ═ F-F0)/F0, F being the fluorescence intensity measured at 520nm for the data in the presence of the target and F0 being the fluorescence intensity measured at 520nm in the absence of the target. Notably, the fluorescence signal of other homologous mirnas is lower compared to the high fluorescence signal triggered by the target miRNA. let-7d, miR-141, miR-15a and miR-200b signals account for 1.09%, 3.69%, 6.48% and 9.01% of miR-121 signals respectively. Therefore, the method has good selectivity and specificity for miRNA detection. In fact, the present biosensor benefits from entropy-driven reactions in so good an ability to discriminate between homologous mirnas. In this system, the thermodynamics and kinetics of the reaction are coupled together, so that the binding free energy (Δ G) of this DNA circuit reaches a dynamic equilibrium. Even a change of one base greatly affects Δ G-reduced hybridization efficiency.

The invention tests the repeatability of the sensing strategy. The method comprises the following steps: three miRNA samples (0.1nM, 1nM, and 10nM) were taken at three concentration levels, each concentration was measured three times, and the relative standard deviation was calculated. The test results are shown in fig. 15, and the relative standard deviations of the miRNA sample to be tested at the above three concentration levels are 4.22%, 3.81%, and 1.05%, respectively. The results show that the sensing strategy obtains more satisfactory repeatability.

In summary, we designed a novel, simple, entropy-driven 3D DNA walking machine and coupled CHA reactions. The biosensor was successfully applied to picomolar miR-21 detection, showing several attractive advantages. First, it is an enzyme-free system, does not require stringent reaction conditions and high detection costs, and eliminates the tedious thermocycling procedure. In addition, the streptavidin-coated polystyrene microspheres are used as the tracks of the 3D DNA walking machine, so that the repeatability is good, and the target is used as random DNA walker, so that the structure of the 3D DNA walking machine is simple. Finally, the composite biosensor has obvious specificity to homologous miRNA, except miRNA-200b, the signal of miR-121 is not more than 5%. Meanwhile, the method obtains satisfactory results in a repeatability test (1.05-4.22%) and a recovery rate test (99.5-104.8%). Therefore, we have reason to believe that this integrated enzyme-free 3D DNA walker-coupled CHA reaction has important application prospects in point-of-care diagnostics.

Drawings

FIG. 1 is a scanning electron microscope image of polystyrene particles coated with streptavidin

FIG. 2 is a spectrofluorimeter profile (scan) of a single entropy driven feasibility analysis

FIG. 3 is an agarose gel electrophoresis of the entropy-driven strand displacement reaction and the CHA reaction

FIG. 4 is a spectrofluorometer pattern (scan) of the feasibility of the 3D DNA walking machine coupled CHA reaction

FIG. 5 is a spectrofluorometric spectrum (kinetics) of the feasibility analysis of the 3D DNA walking machine coupled CHA reaction

FIG. 6 is an optimization of the number of toehold of S

FIG. 7 optimization of F concentration

FIG. 8 optimization of CHA reaction temperature

FIG. 9 optimization of CHA reaction time

FIG. 10 optimization of EDR reaction time

FIG. 11 is the fluorescence signal at the level of miRNA21 at various concentrations

FIG. 12 is a linear relationship between miRNA21 and fluorescence signals at different concentrations

FIG. 13 is a comparison of the present invention with other methodologies

FIG. 14 is a specificity test for a sensing strategy

FIG. 15 is a reproducibility test of the sensing strategy

Detailed Description

Preparing the biosensor based on entropy-driven 3-D DNA walking machine coupling catalysis hairpin self-assembly, and operating according to the following steps:

(1) preparation of 3-D DNA Walking machine

The purified three nucleic acid chains LS, AS, S and TEMg buffer are annealed and then refolded to prepare the LS/AS/S trinucleotide complex (C3). The ratio of LS to AS to S is 1: 1.5. The annealing process was carried out by heating the solution to 95 ℃ for 5min in a PCR instrument and then slowly lowering the temperature to 25 ℃ at a rate of 0.1 ℃ per second. Then 50. mu.L of streptavidin-modified polystyrene microspheres (PS) were centrifuged at 12000 rpm for 5 minutes, washed 2 times with binding buffer and centrifuged to discard the supernatant. Resuspend with 50. mu.L binding buffer and add 5. mu.L. mu. M C3. After incubation in an incubator at 37 ℃ for 15 minutes, the supernatant was discarded by washing 3 times with TEMg buffer and centrifuging at 12000 rpm for 5 minutes. Finally, resuspending with 50. mu.L of TEMg buffer solution, and finally preparing the DNA functionalized 3-D DNA walking machine (C3/PS)

(2) Preparation of Signal Probe

The hairpin probe (H1) and hairpin probe (H2) were annealed and refolded. The solution was heated to 95 ℃ for 5min in a PCR instrument and then slowly lowered to 25 ℃ at a rate of 0.1 ℃ per second. A FAM-labeled signaling probe RepF and a BHQ 1-labeled signaling probe RepQ were hybridized at a ratio of 1: 2 in 1 XTNaK buffer.

(3) Detection of microRNA

Mu. L C3/PS complex, 2. mu.L, 10. mu.M fuel nucleic acid (FS), 33. mu.L of cutsmart buffer, and 10. mu.L of various concentrations of target were incubated for 60 minutes at 37 ℃ in an incubator. Then 40. mu.L of the supernatant was centrifuged and added to a solution containing 5. mu.L of 1. mu. M H1, 2. mu.L of 10. mu. M H2, 10. mu.L of 50nM RepF: RepQ complex and 23. mu.L of 1 XTNaK buffer were reacted for 60 minutes at room temperature in the absence of light. And finally, measuring the fluorescence signal by using a fluorescence spectrophotometer.