An aptamer-based single particle method for sensitive detection of thrombin using fluorescent quantum dots as labeling probes

Author’s Accepted Manuscript An aptamer-based single particle method for sensitive detection of thrombin using fluorescent quantum dots as labeling probes Jinjin Yin, Aidi Zhang, Chaoqing Dong, Jicun Ren www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(15)00398-7 http://dx.doi.org/10.1016/j.talanta.2015.05.034 TAL15626

To appear in: Talanta Received date: 8 February 2015 Revised date: 10 May 2015 Accepted date: 12 May 2015 Cite this article as: Jinjin Yin, Aidi Zhang, Chaoqing Dong and Jicun Ren, An aptamer-based single particle method for sensitive detection of thrombin using fluorescent quantum dots as labeling probes, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.05.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An aptamer-based single particle method for sensitive detection of thrombin using fluorescent quantum dots as labeling probes

Jinjin Yin, Aidi Zhang, Chaoqing Dong* and Jicun Ren*

School of Chemistry & Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China

*Correspondence: Dr. Chaoqing Dong, Prof. & Dr. Jicun Ren, School of Chemistry & Chemical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China Tel: +86-21-54746001 Fax: +86-21-54741297 E-mail address: [email protected] 1

Abstract In this study, an aptamer-based single particle method was developed for the thrombin detection in human serum samples using fluorescence correlation spectroscopy (FCS). In this method, quantum dots (QDs) were used as the fluorescent probes and thrombin-binding aptamer (TBA) was used as molecular recognition unit. When two QDs probes labeled with TBA (QD-TBA1 and QD-TBA2) are mixed in a sample containing thrombin targets, the binding of targets will cause QDs to form dimers (or oligomers) with bigger sizes, which leads to the nearly double increase in the characteristic diffusion time of QDs in the detection volume of FCS. FCS method can detect the change in the characteristic diffusion time of QDs. Firstly, the diffusion and blinking behaviors of QD-TBA probes in the presence of thrombin were investigated by FCS and total internal reflection fluorescence microscopy (TIRFM) imaging system, and the experimental results documented that QD-TBAs were bound together with “one-by-one” structure when thrombin were added into the solution. And then, the assay conditions were optimized in order to improve the sensitivity and specificity of this method. Under the optimized conditions, the linear range of the method is from 5.0 nM to 500 nM of thrombin, and the limit of detection is about 2.6 nM. Finally, this method was applied to homogeneous determination of thrombin in human serum samples.

Keywords: fluorescence correlation spectroscopy (FCS), thrombin, aptamer, quantum dots (QDs), homogeneous determination.

2

Abbreviations

SELEX, systematic evolution of ligands by exponential enrichment; QDs, quantum dots; BSA, bovine serum albumin; EDC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; TBA, thrombin-binding aptamers; BPP, brightness per particles; FCS, fluorescence correlation spectroscopy; TIRFM, total internal reflection fluorescence microscopy; SEC, size exclusion chromatography.

3

1. Introduction Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotides that can bind to a specific target molecule with high affinity and selectivity, including metal ions [1], ATP [2], toxin [3], proteins [4], and cells [5, 6]. Aptamers have been selected from a large random oligonucleotides pool through systematic evolution of ligands by exponential enrichment (SELEX) process. Compared to antibody, aptamers show certain advantages including automated synthesis, easy modification, and good chemical stability during long-term storage. So far, aptamers as recognition units are used

in

analytical

electrochemistry

methods,

[13-19],

which

mainly

absorbance

[20],

include

fluorescence

colorimetric

[21-25],

[7-12], and

electrochemiluminescence (ECL) [26-32] etc. Fluorescence correlation spectroscopy (FCS) is a single particle detection technique using correlation analysis of fluctuations of the fluorescence intensity due to Brownian motion of particles in a small volume (less than 1.0 fL) [33-38]. FCS has been used in study on molecular diffusion, chemical kinetics, molecular interaction in vitro and in vivo, the selection of aptamers and bioassays [39-42]. The sensitivity of FCS method is highly depended on the detection volume and brightness of the used fluorescent probes that can lead to large fluorescence fluctuations [43]. When compared to fluorescent dyes, semiconductor quantum dots (QDs) show several unique photophysical properties in fluorescent analysis. For example, the fluorescent brightness per QD is one order greater than that of fluorescent dyes due to its higher molar extinction coefficients (10-100X that of dyes) [44]. Meanwhile, QDs show less photobleaching and their surfaces can facilely be decorated with aptamer or other biomolecules. To date, QDs have been a very effective complement to fluorescent dyes for many bioassay applications [45]. Thrombin is a multifunctional serine protease, and plays essential role in the coagulation cascade, thrombosis, and haemostasis [46, 47]. The concentration change of thrombin in blood is associated with disease such as Alzheimer disease [48] and acute coronary syndromes [49]. Multiple methods have been developed for thrombin detection, which include fluorescence spectroscopy [11, 50-54], surface enhanced 4

Raman scattering (SERS) [55], and electrochemistry [13, 15, 18]. However, sensitive, simple, and rapid homogeneous assay of thrombin is still needed in clinical diagnosis. In this study, our motivation is to develop a sensitive bioassay of thrombin by combining FCS with QDs labelling and aptamer molecular-recognizing techniques. Different to the reported homogeneous assay methods that based on the fluorescence or extinction sensing [11, 23, 50-54], its principle is based on the diffusion time sensing induced by thrombin binding in FCS. The procedures of our method are shown in Fig. 1. Firstly, QDs were modified with thrombin-binding aptamers (TBA1 and TBA2) that recognizes the exosites of human thrombin. In the assay, when two QDs probes (QD-TBA1 or QD-TBA2) were mixed with thrombin samples, the binding between thrombin and aptamers caused QDs to form dimers (or oligomers) with bigger diameter. It led to the increase in the characteristic diffusion times of QDs probes in the detection volume of FCS. The quantitative analysis of thrombin is based on the relationship between the characteristic diffusion times of QDs probes and thrombin concentrations. This assay was successfully applied for the determination of thrombin level in human serum samples.

2. Material and methods 2.1 Chemicals and Reagents QD655 (Qdot® 655 ITK™ carboxyl QDs with an emission maximum of ~655 nm) was purchased from Life Technologies (U.S.A). Sephacryl high resolution chromatography media (S200) was obtained from GE Healthcare (Sweden). All the aptamers were synthesized and purified through high performance liquid chromatography (HPLC) by Shanghai Sangon Biotechnology (China). The aptamer sequences were [56, 57]: TBA1: 5’-NH2-(CH2)6-TTTTTTTTTTGGTTGGTGTGGTTGG-3’. TBA2: 5’- NH2-(CH2)6-TTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3’. Thrombin was purchased from Shanghai Shize Biological Technology Co. (China). Bovine serum albumin (BSA, >98%) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) was obtained from Sigma-Aldrich Chemical Co. 5

(Milwaukee, U.S.A). Other chemicals and reagents were from Sinopharm Chemical Reagent Co. (Shanghai, China). All chemicals were of analytical grade or better. The dilution buffer was 50 mM borate buffer (pH 8.0). The reaction buffer for QDs and aptamers was 10 mM Tris-HCl buffer (pH7.4), in which 140 mM NaCl, 5 mM KCl and 1 mM MgCl2 were included. All aqueous solutions were prepared with ultra-pure water, which was prepared from the Millipore Simplicity System (Bedford, MA, U.S.A).

2.2 Biological samples Five human serum samples from healthy subjects were provided by Shanghai Jiao Tong University Affiliated Shanghai Xinhua Hospital, and were stored at -20 °C for further use. All experiments were performed in compliance with the relevant laws and institutional guidelines.

2.3 Preparation of QD-TBA Aptamers (TBA1 and TBA2) were linked with QD655 using EDC-mediated condensation. In the preparation, the carboxyl group of QD655 was first activated with 6 μL of EDC (0.25 mg/mL) in 40 μL of dilution buffer for 15 min. Then different concentrations of aptamer (1μM, 2μM, and 10 μM) were added into the activated mixture for two hours at room temperature, resulting in the covalent linking of QDs with the terminal amine group of aptamer. The final concentration of QD655 was 0.2 μM.

2.4 Purification of QD-TBA conjugates SEC and ultrafiltration separations were used to remove the unreacted aptamer and purify the QD-TBA1 or QD-TBA2 probes. SEC was performed on a home-built gel column filled with S200. Firstly, the column was equilibrated with three column volumes of dilution buffer. And then the probes samples were added to the top of the column for separation. After the samples permeated into gel completely, the samples were separated in the gel with the added dilution buffer. Finally, each drop with red 6

fluorescence was collected in separate tubes. The purified product was stored at 4 oC for further use. In the ultrafiltration purification, the QD-TBA probes were purified from the reaction mixtures of QDs and aptamers with ultrafiltration membrane (Microcon YM-50, Millipore, U.S.A) by centrifugation according to the manufacture’s instructions. Briefly, the probes samples were added to the sample reservoir, and then were inserted into the filtrate vial. The vial was centrifuged at 14,000×g for 20 min at 4 oC. After centrifugation, 100 µL of dilution buffer was added to the sample reservoir for dispersion. These above operations were repeated for 1-8 times. Then, the flowthrough was discarded, and the reservoir was placed upside down in a new vial and spun at 1000×g for 3 min. Finally, the purified QD-TBA1 or QD-TBA2 was transferred into a new vial. The product was stored at 4 oC for further use. The concentrations of the purified QD-TBA probes were determined with FCS method.

2.5 FCS and TIRFM measurements FCS measurements were performed on a home-built FCS system constructed on an inverted fluorescence microscope (IX 71, Olympus, Japan) (Figure S1). The schema of the system was similar to that in our previous reported works [58]. Briefly, the 488 nm laser beam from argon ion laser (ILT, Shanghai, China) was attenuated to 40 µW by a circular reflective neutral density filter. Then the laser beam was expanded by a telescope to just fill the back aperture of a 60×/NA1.2 water-immersion objective (UplanApo, Olympus, Japan) and reflected into the objective with a dichroic mirror (505DRLP, Omega Optical, U.S.A). Then, the laser beam was focused in a droplet of sample solution on the coverslip by the objective. The fluorescence was collected by the same objective and passed through the same dichroic mirror and a band pass filter (660DF50, Omega Optical, U.S.A). Finally, it was coupled into a 60 µm pinhole at the image plane in front of single photon counting module (SPCM-AQR14, Perkin-Elmer EG&G, Canada). The fluorescence fluctuations were correlated by a real time correlator (Flex02-12D/C, Correlator.com). 7

Total internal reflection fluorescence microscopy (TIRFM) imaging of QDs samples was performed on TIRFM imaging system constructed on an Olympus IX 71 inverted fluorescence microscope [59]. Samples were excited with 488 nm argon ion laser (ILT laser, Shanghai, China) and the laser power monitored in front of the microscopy objective (60´/NA1.45, Olympus) was about 0.5 mW after it was attenuated. Fluorescence from the sample was collected by the same objective, separated from the excitation light by a dichroic mirror (505DRLP, Omega Optical, U.S.A) and an emission filter (660AF50, Omega Optical, U.S.A), and then focused into EMCCD camera (Evolve 512, Photometrics, U.S.A). Image acquisition and processing were performed using the Micro-manager (Vale Lab, UCSF) and ImageJ software (NIH, U.S.A). All measurements were performed at room temperature. The coverslips (#1, Fisher, U.S.A) for TIRFM imaging were thoroughly cleaned in hot chromic acid mixture, 0.1M NaOH solution, ethanol, and ultrapure water (each for 15 min) and dried in a jet of N2. Then samples were prepared by spin casting QDs samples in 1% Polyethylene Oxide (PEO) (w/w) (MW 1000,000, Sigma-aldrich), and left for 60 s to allow complete drying of PEO.

2.6 Procedure for thrombin detection The thrombin detection procedure is shown in Fig. 1. In the assay, 10 μL of QD-TBA1 (50 nM) and 10 μL of QD-TBA2 (50 nM) were mixed in 200 μL of sterilized tube and then 70 μL of reaction buffer and 10 μL of different concentrations thrombin (from 50 nM to 5 μM) or human serum sample were added. The human serum samples have been diluted ten times with reaction buffer before measurements. The mixture was reacted for 60 min at room temperature. After incubation, the samples were measured on the FCS system. Each measurement time was 120 s, and three repeated measurements were performed. In order to evaluate the specificity of method, the response of diffusion times to different concentrations of added BSA were evaluated under identical experimental procedure to those employed for thrombin. Analytical performance was assessed via linear range, limit of detection (LOD), reproducibility, and specificity. 8

2.7 Data analysis In FCS, the autocorrelation function G(t ) is defined as Eq. (1).

G(t) =

ádI(t)dI(t + t)ñ á I(t)ñ 2

(1)

Where the angular brackets represent a time average, dI (t) the fluorescence intensity fluctuations at a given time t, dI (t+t) fluorescence intensity fluctuations at a given later time and t the time delay. The theory of FCS had been described elsewhere. [33] The diffusion of QDs in an elliptical Gaussian-shaped confocal detection volume can be described by a 3D-diffusion model as Eq. (2). [35]

G(t) =

1 1 1 . . N 1 + t / tD 1 + (wxy / wz )2 × t / tD

(2)

Where N is the average number of nanoparticles in the detection volume, ωxy and ωz is the lateral and axial radii of the detection volume at the e-2 point of the Gaussian laser beam intensity. τD is the characteristic diffusion time of nanoparticles, which is related to the diffusion coefficients of nanoparticles(D). [60]

tD = w2xy / 4D

(3)

The raw FCS data were nonlinearly fitted with the Levenberg-Marquardt algorithm using Microcal Origin 6.0 software package according to Eq. (2). Induced from Eq. (3) and Stokes–Einstein equation (4), the determined τD in FCS is proportional to hydrodynamic diameter (d) of particles or aggregates as shown in Eq. (5). D=kBT/ (3πhd)

(4)

2 tD = 3phwxy d / (4k B T)

(5)

Where kB is the Boltzmann constant, T expresses the temperature, and η is the dynamic viscosity of the solution. In FCS measurements, the brightness per particle (BPP) values of QDs probes are measured as Eq. (6). [61]

BPP = I / N

(6)

9

3. Results and Discussion 3.1 Principle of aptamer-based single particle method for thrombin detection Fig. 1 shows the principle of single particle method for homogeneous detection of thrombin by combining FCS with QDs labels and aptamer molecular-recognizing techniques. The absorption or emission spectra of QD655 are shown in Supplementary Material section (Fig. S2). As shown in Fig. 1b, when different concentrations of thrombin were mixed with QDs probes (QD-TBA1 and QD-TBA2 mixture), the binding reaction between thrombin and aptamers caused QDs to form dimers (or oligomers) with bigger diameter. Therefore, in principle the diffusion times of QDs aggregates in the FCS detection volume (about 0.6 fL) doubly increase with concentration of thrombin. The quantitative analysis of thrombin is based on the established relationship between characteristic diffusion times and thrombin concentrations. [Figure 1]

3.2 Purification strategies of QD-TBA In the preparation of QD-TBA probes, two separation methods (SEC and ultrafiltration) were tried to purify QD-TBA from the reaction mixture of QD-TBA conjugates and unreacted aptamer. The BPP characterization of QDs was proposed to evaluate the separation efficiency of methods. Fig. 2 demonstrates the effects of separation methods on the BPP of QD-TBA probe. It was found that BPP values of QD-TBA were stable before and after the SEC separations, while BPP values were remarkably reduced with the increased ultrafiltration times. According to our previous experiment experiences [62] and the manufacture’s suggestion, more than three repeated ultrafiltration operations were required in order to completely remove the unreacted aptamer. Herein, on comparison, SEC was more suitable for the purification of QD-TBA in our case. [Figure 2] Meanwhile, the normalized autocorrelation curves of QDs and purified QD-TBA with SEC proved that TBA had been successfully linked with QDs. As shown in Fig. 10

S3, these curves of QD-TBA shifted to right compared with that of QDs, which suggested that the hydrodynamic diameter of QDs increased with surface of QDs modified with TBA. In order to verify the recognition ability of QD-TBA on thrombin, different concentrations of thrombin were added into the QD-TBA1 and QD-TBA2 mixtures and reacted in the reaction buffer. The FCS measurement results (Fig. 3A) show that these curves of samples gradually shift to right with the increase of thrombin concentrations, which suggests that the QDs are bound together due to the interaction of thrombin with aptamers. The autocorrelation curves of QDs were fitted by Eq. (2) well, the fitting residuals were all lower than 0.1, and the correlation coefficients (R2) were 0.982(as shown in Fig. 3A). Meanwhile, the relation between the logarithm of characteristic diffusion time (log τD) and the logarithm of thrombin concentration (log C) shows a good linearity (Fig. 3B). [Figure 3]

Furthermore, TIRFM imaging characterization also showed the binding of QDs with “one-by-one” structure induced by thrombin. On one hand, white dots with bigger size of light spot were observed in the TIRFM imaging of the QD-TBA samples with thrombin added (Fig. 4B) compared with that in that of the QD-TBA samples without thrombin added (Fig. 4A). The main reason is that the aggregates of QDs induced by thrombin will emit strong fluorescence. On the other hand, Fig. 4C is the typical fluorescence trajectory of single white light spot extracted from the imaging movie of QD-TBA probes without thrombin, which expresses the unique blinking property of individual QDs. On comparison, Fig. 4D is the typical fluorescence trajectory of single white light spot extracted from the imaging movie of the reaction sample of thrombin and QD-TBA probes. Obviously, the blinking property of QDs completely disappeared due to self-binding of QDs. Particularly, amplitude of fluorescence trajectory of blinking dot (Fig. 4C) were only about 50% of non-blinking dot (Fig. 4D), which clearly indicated that two dots were bound together with “one-by-one” structure. 11

[Figure 4]

3.3 Optimization of experimental conditions In order to increase the sensitivity of the assay, the effects of certain factors such as molar ratio of QDs and TBA, QD-TBA concentrations and reaction time were studied. The effects of molar ratio of QDs and TBA on the assay are shown in Fig. 5A. In the different molar ratios, the characteristic diffusion time of QDs greatly increased with the increase of thrombin concentration. In this case, the slope of the curves significantly increased with the increase of molar ratio as well, which indicated that the assay was more sensitive at higher molar ratio (1:5). Therefore, the 1:5 of molar ratio of QDs and TBA was chosen in the following experiments. Fig. 5B shows the effects of QD-TBA concentrations on the assay. It was observed that the characteristic diffusion time of QD-TBA increased with thrombin concentration in the presence of different concentrations of QD-TBA (from 2 nM to 25 nM). In principle, when the thrombin–TBA binding constant is large enough, the lower concentration of QDs should be benefited for enhancement of sensitivity, which is in line with our experimental results that the slopes of the curves decreased with the increase of QD-TBA concentration (5 nM, 10 nM and 25 nM). However, as shown in Fig.5B, the result illustrated that the sensitivity of the assay at the concentration of QD-TBA of 2 nM was not better than that at the concentration of QD-TBA of 5 nM. This phenomenon is mainly attributed to the affinity of the aptamer to thrombin (Kd ~0.5 nM). As a result, 5 nM of QD-TBA concentration was used in subsequent experiments. [Figure 5]

The effects of reaction time on the assay were investigated. As shown in Fig. S4, the characteristic diffusion time of QD-TBA and thrombin mixture increased with reaction time. The result illustrated that the binding between QDs induced by thrombin reached the maximum and remained constant within about 60 min. In the follow-up experiments, the reaction time was fixed at 60 min. 12

3.4 Assay of thrombin in human serum samples and the method specificity Under the optimized experimental conditions above, an aptamer-based single particle method was developed for homogeneous bioassay of thrombin in human serum samples. Fig. 6 reflected the good linear relationship between the logarithm of characteristic diffusion time of aggregates (log τD) and the logarithm of thrombin concentration (log C). The calibration curve of thrombin has a linear working range over two orders of magnitude (from 5.0 nM to 500 nM). The limit of detection (LOD) is estimated to about 2.6 nM based on the average characteristic diffusion time of the blank (the control experiments) plus three times the standard deviation. Measurements were repeated five times to evaluate the reproducibility of the method. The result showed an average of 16% CV, calculated as the mean of all the concentrations tested. Specificity is an essential criterion for any analytical methods. In order to confirm the binding specificity of the aptamer to the thrombin, different concentration of BSA were used to replace the thrombin. The whole experiment procedure was same to those of thrombin assay. As shown in Fig. 6, the weak response of the characteristic diffusion time versus different BSA concentration was observed. It indicated that non-specific interactions were much weaker compared with the specific binding and thus can be neglected. The influence of other proteins such as Hb and IgG on measurements can be found in the Figure S5. The highly specific and high-affinity interaction between thrombin and TBA allows us to discriminate against nonspecific binding and thus readily detect thrombin even in human serum. In order to demonstrate the feasibility of the method, this method was applied for the direct determination of thrombin in human serum. In the assay, all the serum samples were diluted 10 times with reaction buffer, and the results are shown in Table 1. The concentrations of thrombin in the different human serum samples are determined in the range of 20 nM and 100 nM, the RSDs (between 1.7% and 6.5%), and the recoveries of the thrombin (between 80% and 110%) are acceptable. The results illustrated that the method had promising sensing abilities even in complex biological samples. 13

[Figure 6] [Table 1] Compared with the other reported methods, the principle of this method is different and has a comparable LOD (Table 2), although it is still lower than that for some electrochemical methods. In addition, the method can allow thrombin detection in serum. [Table 2] 4. Conclusions In this work, a single particle method for the homogeneous, sensitive, and specific detection of thrombin has been developed by combining FCS with advantages of QDs and aptamer. Firstly, the preparation procedure of QD-TBA probes was investigated, and BPP characterization was proposed to assess the performances of different purification methods. The results documented that SEC was well suitable for the purification of QD-TBA. And then, the experimental conditions were optimized, and the quantitative relationship between the characteristic diffusion time and thrombin concentration was investigated. Finally, this method was successfully applied for the determination of the thrombin levels in human serum samples. The high sensitivity and good specificity of the method were attributed to the unique properties of QDs and aptamers. This new method can be extended to the sensitive detection of other target molecules when new aptamers were screened with SELEX and higher brightness of probes was prepared. Supporting Information Additional information about photoluminescence (PL) spectra and UV-vis spectroscopy of QD655, FCS and their fitting residual curves of QDs, QD-TBA1 and QD-TBA2, effect of reaction time on binding reaction, the specificity of the method.

ACKNOWLEDGEMENTS This work was financially supported by NFSC (21135004, 21327004, and 21475087), Innovation Program of Shanghai Municipal Education Commission 14

(14ZZ024), and SMC-Chenxin Young Scholar project sponsored by Shanghai Jiao Tong University.

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Table 1. Assay results of thrombin in real human serum (n=3).

Table 2. Comparison of aptamer-based homogeneous methods for the detection of thrombin.

20

Figure Captions

Fig. 1. The procedure of aptamer-based single particle method for homogeneous detection of thrombin. QDs were linked with aptamer (TBA1 and TBA2) using EDC as coupling reagent (a). The QD-TBA probes reacted with the different concentration of thrombin or human serum samples and formed the dimer (b). The schematic diagram of change of characteristic diffusion time with thrombin concentration was measured by FCS (c).

Fig. 2. Effect of QDs purification methods. The reaction molar ratio of QDs and TBA was 1:5. The sampling time was 120 s.

Fig. 3. (A)The normalized autocorrelation curves, their fitting curves and their fitting residuals curves of QD-TBA with different concentration of thrombin (a-0 nM, b-5.0 nM, c-25 nM, d-50 nM, e-100 nM, f-500 nM). (B) The linear relationships between the logarithm of their characteristic diffusion time (log τD) and logarithm of thrombin concentration (log C). The concentration of QD-TBA1 and QD-TBA2 were 5.0 nM. The sampling time was 120 s. The error bars represent the standard deviation from three repeated measurements.

Fig. 4. TIRFM images of QD-TBA probes without thrombin (A) or QD-TBA probes incubated with 50 nM thrombin (B). These samples were dispersed in 1% PEO matrix on the glass coverslip. The exposure time for per image was 60 ms. Figure (C) and (D) were typical fluorescence intensity trajectories of individual bright spot from their imaging movies, respectively. The black lines as background signal were from blank spot without bright spot included in the whole movie sequences.

Fig. 5. (A) Effect of molar ratio of QDs and TBA on the sensitivity. The concentration of QD-TBA1 and QD-TBA2 were 5.0 nM, respectively. (B) Effect of QD-TBA 21

concentration on the sensitivity. Here the molar ratio of QDs and TBA was 1:5. The reaction and measurements were performed at room temperature. The sampling time was 120 s. The error bars represent the standard deviation from three repeated measurements.

Fig. 6. Response of characteristic diffusion times before (the control sample) and after different concentrations of thrombin added, and their comparison with that after BSA added. The molar ratio of QDs and TBA was 1:5. The concentrations of QD-TBA1 and QD-TBA2 were 5.0 nM, respectively. The sampling time was 120 s. The error bars represent the standard deviation from three repeated measurements.

●A novel single nanoparticle detection method based on FCS was reported. ●A homogeneous assay of thrombin was developed using this method. ●Aptamer and QDs were used as recognition unit of thrombin and probes. ●The method can detect the changes of diffusion times before and after reaction. ●The method is used for detection of thrombin in human serum.

22

*Graphical Abstract (for review)

Table 1

Table 1. Assay results of thrombin in real human serum (n=3). Sample

Thrombin in samples (nM)

RSD (%)

Thrombin added (nM)

Thrombin found (nM)

RSD (%)

Recovery (%)

1

3.25

2.4

25.0

29.3

1.7

104.2

2

2.69

2.2

50.0

42.8

4.6

80.2

3

3.86

3.1

50.0

50.6

2.4

93.5

4

8.30

3.7

50.0

56.5

5.9

96.4

5

9.61

2.3

100.0

117

6.5

107.4

Table 2

Table 2. Comparison of aptamer-based homogeneous methods for the detection of thrombin Method

mechanism

Signal output/ reporter

LOD

Reference

QDs aptamer beacons fluorescence aptasensor fluorescence aptasensor

fluorescence recovery with quencher released with thrombin binding fluorescence quenching with thrombin binding fluorescence restoration with the displacing of FAM-DNA from SWCNH with thrombin binding fluorescence restoration with the displacing of FAM-DNA from SWCNT with thrombin binding photoluminescence enhanced by aggregation of silver nanoparticles with thrombin binding changes in the absorption ratio with thrombin binding diffusion time increase with thrombin binding

fluorescence, QDs fluorescence, sliver nanoclusters fluorescence, FAM

1 µM

[50]

1 nM

[51]

100 pM

[52]

fluorescence, FAM

1.8 nM

[53]

fluorescence, silver nanoparticles

3.1 pM

[54]

colorimetric/absorbance, Gold nanoparticles diffusion time, QDs

0.83 nM

[23]

2.6 nM

In this work

fluorescence biosensors two-photon biosensing colorimetric sensing FCS

FAM: carboxyfluorescein; SWCNH: Single-walled carbon nanohorns; SWCNT: single-walled carbon nanotube

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 5A

Figure 5B

Figure 6