Fluorescence Detection of H5N1 Virus Gene Sequences Based on

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Fluorescence Detection of H5N1 Virus Gene Sequences Based on Optical Tweezers with Two-photon Excitation Using a Single NIR Nanosecond Pulse Laser Cheng-Yu Li, Di Cao, Ya-Feng kang, Yi Lin, Ran Cui, Dai-Wen Pang, and Hong-Wu Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00065 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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Analytical Chemistry

Fluorescence Detection of H5N1 Virus Gene Sequences Based on Optical Tweezers with Two-photon Excitation Using a Single NIR Nanosecond Pulse Laser Cheng-Yu Li,† Di Cao,† Ya-Feng Kang, Yi Lin, Ran Cui, Dai-Wen Pang and Hong-Wu Tang* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, 430072, People’s Republic of China

_________________________ *Corresponding

Author:

E-mail:

[email protected].

Phone:

+86-27-68754067. †

These authors contributed equally to this work. 1

ACS Paragon Plus Environment

+86-27-68756759.

Fax:


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Abstract: We present an analytical platform by combining near-infrared optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences. A heterogeneous enrichment strategy, which involved polystyrene (PS) microsphere and quantum dots (QDs), was adopted. The final hybrid-conjugate microspheres were prepared by a facile one-step hybridization procedure by using PS microspheres capturing target DNA and QDs tagging respectively. Quantitative detection was achieved by the optical tweezers setup with a low-cost 1064 nm nanosecond pulse laser for both optical trapping and two-photon excitation for the same hybrid-conjugate microsphere. The detection limits for both neuraminidase (NA) gene sequences and hemagglutinin (HA) gene sequences are 16 ~ 19 pM with good selectivity for one-base mismatched, which is approximately one order of magnitude lower than the most existing present fluorescence-based analysis method. Besides, due to the fact that only signal from the trapped particle is detected upon two-photon excitation, this approach showed extremely low background in fluorescence detection and was successfully applied to directly detect target DNA in human whole serum without any separation steps and the corresponding results are very close to that in buffer solution, indicating the strong anti-interference ability of this method. Therefore, it can be expected to be an emerging alternative for straightforward detecting target species in complex samples with a simple procedure and high-throughput.

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Optical tweezers (OT)

1, 2

, which is traditionally formed by a tightly focused Guassian laser beam,

has been demonstrated to be one of the most effective micromanipulation technology in material science and life science due to its advantages of non-contact and non-invasion. To investigate various physical and chemical properties of a single particle, OT can act as a powerful fixed handle to make it possible for a long term observation, thus it has been widely used in trapping and manipulation of microscale and nanoscale objects, such as red blood cells3, 4, T cells5, single proteins6, 7, polymer chains8, 9 carbon nanotubes10, 11, gold and silver nanoparticles12-14, upconversion nanoparticles15, 16, quantum dots17-20, and so on. Besides, owing to pico-Newtons of mechanical forces produced by OT, this fascinating technology can also be expended to study various biological issues, including DNA melting21, cell-cell interaction22, DNA-protein interaction23, movement of motor proteins24, etc. However, this unique single-particle based approach has not attracted wide attentions in the field of analytical sciences yet.

Two-photon excitation (TPE) was first experimentally proved by Garrett et al.25 and was applied in cell imaging by Webb’s group in 199026. Different from single-photon absorption, TPE is a nonlinear process and occurs in a molecule absorbs two photons simultaneously, which is normally induced by extremely high instantaneous photon densities with a pulse laser beam. Due to a near-infrared (NIR) laser beam being used as the excitation source and tightly converged, TPE involves many incomparable advantages, including deeper tissue penetration, higher spatial resolution, lower background fluorescence, and less photodamages27, 28. Thus, under the condition of TPE, various kinds of targets, such as DNA, proteins, virus, etc. can be detected in complex circumstances without the interference of other impurities through fluorescence-based analysis. Compared with the 3



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conventional organic dyes, quantum dots (QDs) have excellent optical properties, relating to broader excitation spectrum, narrower emission spectrum,

higher quantum

yield and

stronger

anti-photobleaching ability29-31. Additionally, according to the available literatures, the TPE cross sections of these semiconductor nanomaterials achieve as high as nearly 105 GM32, 33 (1GM = 10-50 cm4·s·molecule-1·photon-1), which is larger than any other reported fluorescent labels. Apparently, QDs can be an outstanding candidate for labeling in TPE experiment.

During the past decades, detection of DNA has become a hot topic for scientists of various subjects, ascribed to its importance in gene sequencing, biomedical research, diagnosis and treatment for virus, and so on34-36. To improve the detection sensitivity for practical applications, many kinds of amplification methods have been established, including polymerase chain reaction (PCR)37, hybridization chain reaction (HCR)38, rolling circle amplification (RCA)39, etc. In addition, progresses have been made in ultrasensitive fluorescence-based methods for nucleic acid detection towards amplification-free and the concept of single molecule detection has been introduced40. Unfortunately, the analysis of complex biological and medical samples such as whole serum remains a great challenge.

As is well known, H5N1 virus, which is spread by the migration of wild birds, is one of the highest lethality for human beings in the family of avian influenza virus (AIV)41. Generally, the surface of H5N1 virus exist two types of glycoproteins, namely neuraminidase (NA) and hemagglutinin (HA), making the virus has a strong ability to infect host cells. In this paper, we proposed a novel analytical platform based on optical tweezers with two-photon excitation for fluorescence detection of NA and 4


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HA gene sequences of H5N1 virus. The detection principle of this approach is illustrated in Scheme 1. Carboxylated polystyrene microspheres (CPSs) are used as the solid phase carrier and QDs are selected as the fluorescent tag. By measuring the fluorescence intensities (quantified by gray-level) of 50 microspheres in each concentration group, the average value is used for quantitative detection of H5N1 gene sequences. It is worth noting that OT plays an important role in this experiment, by which the individual trapped microspheres are always fixed at the trapping center (the focal point) to ensure the same excitation conditions. To reduce the expenditure of laser device in performing TPE, a low-cost Q-switched nanosecond pulsed laser was used as the light source for both OT and TPE excitation instead of a conventional femtosecond pulsed laser. Simultaneously, in order to improve the detection sensitivity and avoid tedious amplification process for DNA sensing, an Electron-Multiplying CCD (EMCCD) is coupled to this setup and a heterogeneous enrichment method is adopted by using polystyrene microspheres capturing target molecules. To better reflect the strengths of this single-microsphere based detection approach, we investigated the detection capability of this method in buffer solution and human whole serum, respectively. The results demonstrated that the target DNA can be sensitively detected with strong anti-interference ability and good selectivity. To the best of our knowledge, this work paved a new way to the application of optical tweezers in analytical science.

EXPERIMENTAL SECTION Materials and Reagents. Carboxylated polystyrene microspheres (3.0 - 3.9 µm) was purchased from Aladdin Industrial Inc. (China). Streptavidin-modified 605 nm CdSe/ZnS QDs (quantum yield 85%) and streptavidin-modified 705 nm CdSeTe/ZnS QDs

(quantum yield 52%) were supplied by

5



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Wuhan Jiayuan Quantum Dots Co. (China). 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

(EDC·HCl,

99%),

2-(N-Morpholino)ethanesulfonic

acid (MES,

99%) and

Tris(hydroxymethyl)aminomethane (Tris-HCl, 99%) were obtained from Sigma-Aldrich Co. (USA). Other analytical grade reagents used without further purification were provided by Sinopharm Chemical Reagent Co. (China). All the DNA oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. (China) with the following sequences: Amine-modified capture DNA sequences for NA and HA: C-NA: 5/-NH2-(CH2)12-ATGAAGCCTGCC-3/ C-HA: 5/-NH2-(CH2)12-AGGAAAAGATCT-3/ Biotinylated probe DNA sequences for NA and HA: P-NA: 5/-CATTACTTGGTCC-(CH2)3-biotin-3/ P-HA: 5/-TCTTGGTTGGTATT-(CH2)3-biotin-3/ Target sequences of NA and HA: T-NA: 5/-GGACCAAGTAATGGGCAGGCTTCAT-3/ (Genbank AF509096.1) T-HA: 5/-AATACCAACCAAGAAGATCTTTTCCT-3/ (Genbank AF509020.1) Mismatched sequences of NA and HA: MT1-NA: 5/-GGACCAAGTAATCGGCAGGCTTCAT-3/ MT2-NA: 5/-GGACCAAGTAATCCGCAGGCTTCAT-3/ Random-NA: 5/-AATGCTACATTTTGCCATCGATTAC-3/ MT1-HA: 5/-AATACCAACGAAGAAGATCTTTTCCT-3/ MT2-HA: 5/-AATACCAACGTAGAAGATCTTTTCCT-3/ Random-HA: 5/-CCCGTATTTAAGCTATTAAGCGATA-3/ 6


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Apparatus. A UV-2550 spectrophotometer (Shimadzu, Japan) was used to measure UV-vis absorption spectra. Fluorescence images were recorded by a Nikon ECLIPSE TE2000-U inverted fluorescence microscope equipped with a CCD. Agarose gel electrophoresis was performed by a voltage type electrophoresis apparatus equipped with an imaging system (Beijing Liuyi Co., China).

Construction of the optical tweezers setup Our home-built optical tweezers setup was based on an inverted microscope (Olympus IX70, Japan) with an infinity-corrected optical system as shown in Fig. 1. A 1064 nm diode pumped passively Q-switched solid state laser (CryLas, Germany) with pulse duration ≤ 1.5 ns and repetition rate 10 kHz was simultaneously applied as the trapping laser source and the excitation source for two-photon excitation. The laser beam was first passed through an absorptive neutral density filter (NDF, Zolix, China) to reduce its mean power at the sample to dozens of milliwatt for optimal trapping, and then reflected by two adjustment mirrors (M1 and M2, Zolix, China). Afterwards, the laser beam was expanded 5-fold by a beam expander system, which was composed of two lens with different focal lengths (L1: 25 mm and L2: 125 mm, Zolix, China), to slightly overfill the back aperture of the microscope objective. Finally, the laser beam was reflected upward by a dichroic mirror (850 nm, Edmund) and tightly focused by a 100 × oil immersion objective (NA = 1.30, Olympus, Japan) into the sample chamber to form a diffraction limited spot with 1 µm in diameter. A short-pass filter (FF01-760/SP-25, Semrock) is used as the barrier filter and positioned in the filter cube. The two-photon fluorescence signals originated from the trapped hybrid-conjugated microspheres and the real time images were detected and captured by a cooled EMCCD (Photometrics, Canada). 7



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Additionally, a white-light LED lamp focused by a lens was used as the bright-field illumination. The mean laser power passed through NDF was approximately 130 mW, which was measured by a near-infrared power meter (Coherent, USA). The corresponding mean power reached the sample chamber was about 70 mW. Consequently, the instantaneous power density at a trapped microsphere was approximately 5.9 × 1011 W/cm2. According to literature26, such a high power density was enough to realize two-photon excitation.

Preparation of capture DNA functionalized polystyrene (PS) microspheres C-NA was covalently conjugated to the surface of CPSs through EDC chemistry with a slight modification42. Before conjugating, a certain amount of CPSs were washed twice with 100 µL of MES buffer solution (10 mM, pH 5.0). Subsequently, 0.3 nmol C-NA and 5.0 µL of 10 mg/mL EDC were added into the as-washed CPSs, and then incubated for 1 h at room temperature with gentle shaking. Afterwards, 2.0 µL of 100 mg/mL EDC was added into the solution to increase the coupling efficiency and continued to incubate overnight with gentle shaking. Finally, the as-prepared microspheres were washed three times and resuspended in 200 µL of hybridization buffer (50 mM Tris, pH 8.0, 200 mM NaCl) for further use. The immobilization of C-HA onto the surface of CPSs was followed the same procedure.

Quantification of capture DNA on the surface of CPSs Various volumes of 10 µM of C-NA were diluted to 500 µL to keep the final concentration ranging from 0 to 1.2 µM, and then their corresponding UV-vis absorption spectra were measured with the slits set at 2 nm. The UV-vis absorption value at 260 nm, which is the characteristic absorption peak 8


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of DNA, was plotted as a function of the concentrations of C-NA. By measuring the absorbance of the centrifugal supernatant at 260 nm, the molar number of residual C-NA can be quantitatively calculated from the regression equation of standard C-NA. As a result, the actual reaction amounts were equal to the difference value between the added quantity and the residual quantity. Quantification of C-HA on the surface of CPSs was followed the same procedure.

Preparation of QD probes Preparation of 605 nm QD probes was based on the well-known high specificity and affinity interaction between streptavidin and biotin43. Briefly, 40 µL of 1 µM streptavidin-modified 605 nm CdSe/ZnS QDs was first mixed with 3 nmol P-NA in 400 µL of 0.01 M PBS buffer solution (pH 7.2) and then incubated for 1 h at room temperature with gentle shaking. The excessive P-NA was removed by spin filtration in Millipore Microcon 50,000 molecular weight cut-off spin filters with centrifuging twice at 10000 r/min for 5 min and the final QD probes were resuspended in 100 µL of hybridization buffer for further use. Preparation of 705 nm QD probes was followed the same principle.

Quantification of probe DNA on the surface of QDs Agarose gel electrophoresis was employed to verify whether the probe DNA was successfully linked onto the surface of QDs. Various molar ratios (1:1, 1:2, 1:5, 1:8, 1:10, 1:20) between P-NA and the streptavidin-modified 605 nm CdSe/ZnS QDs was mixed and incubated for 1 h at room temperature with gentle shaking. After removing the unreacted P-NA, 5 µL of each as-prepared QD probes contained 0.5 µL of glycerin were loaded in 1% agarose gel and then placed in 0.5× TAE buffer 9



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solutions, in which pure QDs solution was loaded in another groove as the control group. After running under the voltage of 90 V for 40 min, their mobility distance was positioned by a UV lamp and the corresponding digital images were recorded by a gel imaging device. Quantification of P-HA on the surface of 705 nm QDs was followed the same procedure.

Construction of hybrid-conjugated microspheres Construction of hybrid-conjugated microspheres was carried out by a facile one-step hybridization procedure, which was performed by mixing the capture DNA modified PS microspheres, the target DNA (NA or HA) and the QD probes. In a typical experiment, 3 pmol target DNA (NA or HA) dissolved in 200 µL of hybridization buffer was mixed with 20 µL of microsphere carriers and 4 µL of QD probes. Afterwards, the mixture was incubated for 6 h at 37 °C with gentle shaking. Finally, the hybrid-conjugated microspheres were washed three times and resuspended in 100 µL of hybridization buffer for further quantitative detection.

Optical trapping and manipulation of a single hybrid-conjugated microsphere The above hybrid-conjugated microspheres were first sonicated for approximately 2 min and then vortexed three times to ensure its monodispersity. Then they were diluted ten-fold so that the movement of a trapped microsphere will not be disturbed by its surrounding microspheres. During trapping experiment, a closed sample chamber was used to prevent the medium from volatilizing too fast due to laser thermal effect. Therefore, the chamber with deepness ~0.5 mm was made on a cover glass using vacuum silicone grease, and then 10 µL of the microspheres were added into it with another cover glass covering on the chamber. The horizontal manipulation of a trapped 10


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hybrid-conjugated microsphere was performed with a passive manipulation method, by which the trapping microsphere was captured while the sample chamber was moving slowly (Fig. 3SA-Fig. 3SC). The longitudinal movement of a trapped microsphere was achieved through an active manipulation method, in which the beam position was moved by adjusting the objective in axial direction (Fig. 3SD).

Quantitative detection of NA and HA gene sequences In order not to make the two-photon fluorescence signals supersaturated during quantitative detection and reach the best signal-to-noise ratio simultaneously, the appropriate choice of exposure time and gain is of significance. For nM region of NA and HA genes, we acquired the fluorescence signals by using an exposure time of 100 ms and 150 ms with electron multiplier gain 200, respectively. For pM region of NA and HA genes, we acquired the fluorescence signals by using exposure time of 800 ms and 1000 ms with electron multiplier gain 300, respectively. Finally, we measured the fluorescence intensities of 50 microspheres in each group and the average intensity was used for quantitative analysis.

Quantitative detection of NA and HA gene sequences in human whole serum The experimental steps are similar to that in buffer solution. Except that, during the hybridization procedure, human whole serum containing target DNA (NA or HA) was mixed with the microsphere carriers and the QD probes. The final hybrid-microspheres were resuspended in human whole serum for quantitative detection.

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Selectivity investigation 3 pmol target DNA (NA or HA) and the same amount of other mismatched DNA sequences were added into the same hybridization system, respectively. Next, the corresponding mean fluorescence intensities were measured.

RESULTS AND DISCUSSION The amount of capture DNA on the surface of CPSs Due to the strong scattering signals of CPSs in microscale, it is difficult to accurately determine its UV-vis absorption spectra. Therefore, an indirect measurement was applied for quantitative analysis. According to the size and the density (1.047 g/cm3) of CPSs，the molar number of CPSs can be estimated with the following equation: n(mol) = 3m/4πr3ρNA, where m is the mass of CPSs and r is the radius of CPSs, NA is Avogadro's constant and ρ is the density of CPSs, respectively. For 250 µg of CPSs, we calculated its molar number is 2.8 × 10-17 mol. Based on the UV-vis absorption calibration curves of C-NA and C-HA (Fig. S1A and Fig. S1B) and the UV-vis spectra of the centrifugal supernatants (Fig. S1C and Fig. S1D), the molar number of residual capture DNA was estimated to be 0.05 nmol for both cases. Hence, we concluded that a single CPS is conjugated with 8.9×106 DNA molecules. The result is coincident with the protocol of the manufacturer, in which per particle has 3.0×107 carboxyl groups.

Quantification of probe DNA on the surface of QDs According to Parak et al.44, when DNA is attached on the surface of QDs, the surface charge of QDs and the particle size will be changed concurrently. So, these two factors will synthetically influence 12


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the gel electrophoresis velocity of the conjugated QDs. As shown in Fig. S2A, with the increasing molar ratio between 605 nm QDs and P-NA, the mobility distance of DNA-QDs gradually increases, which is ascribed to that the conjugated QDs have more negative surface charge. When the molar ratio is 1:10, the mobility distance of DNA-QDs reach a plateau, indicating that the surface of QDs is fully capped with P-NA. Accordingly, we chose the molar ratio 1:10 as the optimal experimental condition. The result of 705 nm QDs is similar to 605 nm QDs (Fig. S2B), which is attributed to the same number of streptavidin (2-3 streptavidins per particle) modified on the surface of both two commercial QDs.

Characterization of TPE To further demonstrate that fluorescence emission from the QDs-tagged microspheres is realized by TPE, we measured the fluorescence intensity of a trapped microsphere as a function of the mean laser power on sample. Because photobleaching was not obvious and in order to ensure the correctness of the results, the TPE of the same trapped microsphere tagged with either 605 nm or 705 nm QDs was detected continuously within 40s by gradually adjusting the power of the laser. As shown in Fig. 2A and Fig. 2B, the both curve fittings confirm a pure square law dependence of the fluorescence intensity on the mean laser power with a regression coefficient of 0.9892 for 605 nm QDs and 0.9898 for 705 nm QDs, respectively, indicating that a nonlinear absorption process occurred. According to Liu et al.45 , the fluorescent photon generate rate in the process of TPE can be predicted from the following equation: dp/dt = QNδF2, where Q is the quantum yield of the fluorescent tag and N is the number of molecules, δ is the two-photon cross section and F is the photon density, respectively. Theoretically, the order of magnitudes of the fluorescent photon 13



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generate rates were estimated to be 1018 photons per second for the two cases in this work, which is far beyond the existing organic dyes26, 45.

Investigation of TPE fluorescence photobleaching of QDs on a PS microsphere The possibility of photobleaching for a hybrid-conjugated microsphere during optical trapping using the nanosecond pulse laser with such a high instantaneous power is of importance for fluorescence-based analysis. To address this concern, two kinds of microspheres tagged with different QDs were chosen as the research objects. As shown in Fig. 2C, even if the laser irradiation continued 45 s, the trapped microsphere tagged with 605 nm QDs emitted stable fluorescence and no photobleaching was exhibited, whereas the trapped microsphere tagged with 705 nm QDs began to decay slightly after being irradiated for 35 s. The phenomenon is probably a result from the different base material and crystal structure between visible QDs and NIR QDs46. Because the measurement time for each trapped microsphere was less than 1s, the influence of photobleaching is negligible during fluorescence detection.

Quantitative Detection of NA and HA genes in buffer solution For detection of NA genes, 605 nm QDs were used as the fluorescent tag. When the exposure time was 100 ms, the linear relationship between the fluorescence intensity and the concentration of T-NA ranging from 0.5 to 30 nM was obtained as shown in Fig. 3A and the corresponding real-time images captured by EMCCD are shown in Fig. 3C. When the exposure time is increased to 800 ms, the images for 0.5 nM DNA can be clearly captured by EMCCD as shown in Fig. 3D, in which the blank signal (no target DNA is present) was produced by the nonspecific adsorption of QDs to the 14


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microspheres. The linear relationship in the range of 0 pM to 300 pM was investigated as shown in Fig. 3B. The coefficient variations of 50 microspheres in each group are between 4% and 10%, demonstrating the fluorescence uniformity of the hybrid-conjugated microspheres, which is consistent with Fig. 4A. The well linear relationships were probably benefited from two factors. On one hand, the nanomaterials with favorable monodisperse and good water solubility were used as the fluorescent tag, which guaranteed the thermodynamic stability of this reaction system to some extent. On the other hand, the EMCCD responses sensitively and stably. The detection limit, which was calculated according to 3σ/k criterion, where σ is the standard deviation of the blank signal (n =11) and k is the slope for the low concentration range of the linearity, was estimated to be 19 pM in buffer solution.

For detection of HA genes, 705 nm QDs were used as the fluorescent tag. Although same amount of QDs were attached on these two cases of microspheres (30 nM targets are present) in theory, in which per hybrid-conjugated microsphere contains ~1×105 QDs, the fluorescence intensity of a single microsphere enriched with T-HA is distinctly lower than that of T-NA (Fig. 4), which is presumably attributed to the lower quantum yield of 705 nm QDs. For this reason, the optimum exposure time for high concentration groups and low concentration groups were 150 ms and 1000 ms, respectively. The corresponding two calibration curves are shown in Fig. 5A and Fig. 5B, in which the coefficient variation of 50 microspheres in each group is also in the range of 4 − 10%. The images captured by EMCCD are shown in Fig. 5C and Fig. 5D. The detection limit for HA gene is calculated as 16 pM in buffer solution.

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Quantitative detection of NA and HA gene sequences in human serum Since TPE has been best known for its extremely low background fluorescence signals in complex samples, hybrid-conjugated microspheres resuspended in human whole serum were measured without any separation steps to verify that the as-proposed method has a powerful anti-interference ability. We first took photos of the trapped microspheres with a digital camera in 3 s exposure time to investigate whether the complex matrix in human whole serum will produce background fluorescence under TPE. Comparing with the microspheres (30 nM targets are present) in buffer solution (Fig. 6A and 6C), the same microspheres in human whole serum (Fig. 6B and 6D) exhibit no obvious background fluorescence for both NA and HA genes detection within such a high exposure time. Additionally, the background signal intensities were measured as approximately mean gray-level 216 for NA and mean gray-level 264 for HA in serum. These values were almost equal to that in buffer solution (mean gray-level 214 for NA and mean gray-level 267 for HA), proving that the background signal from the complex biological fluid is negligible owing to TPE. Moreover, the fluorescence intensity of the trapped microspheres in serum is very close to that in buffer solution as shown in Fig. 6, demonstrating the reliability of the heterogeneous enrichment strategy using PS microspheres. As can be seen from the detection results shown in Fig. S4 and Fig. S5, the linear relationships between the fluorescence intensity and the concentration of targets are still favorable without pronounced changes and the detection limits for NA genes and HA genes are 16 pM and 18 pM, respectively, which are quite similar to that in the buffer system. Although real samples were not analyzed with this system, these results indirectly illustrates that our approach is applicable in complex samples with tiny interference signals caused by impurities.

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Selectivity investigation To evaluate the selectivity of this method for DNA detection, the fluorescence intensities of the one-base mismatched DNA sequences, two-base mismatched DNA sequences, and random DNA sequences were measured following the same procedure of the completely complementary DNA sequences. As shown in Fig. 6E, the fluorescence intensities of one-base mismatched DNA sequences for both NA and HA genes decreased by nearly 65%. For two-base mismatched DNA sequences, the fluorescence intensities are significantly lower than that of the completely complementary DNA sequences, and only take up approximately 35% of the latter. Besides, signals can hardly be detected for the random sequences. The results prove that our analytical platform has good selectivity for DNA detection, by which even one-base mismatched sequence can be markedly discriminated.

Comparison with other fluorescence-based analysis method for DNA To the best of our knowledge, our proposed approach is superior to the most existing fluorescence-based analysis method as shown in Table 1. Although single-molecule detection of DNA is possible53, the analysis of complex samples remains a major concern.

Table 1. Comparison with other fluorescence-based analysis method complex sample method

detection limit /amplification

ref

silver nanoclusters based FRET

500 pM

no/no

47

silica nanoparticles as solid phase carrier

170 pM

no/no

48

using a conjugated cationic polymer to amplify

40 pM

no/yes

49

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optically encoded multifunctional nanospheres

87 pM

as fluorescent tag

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yes (with separation) 50 /no

magnetic microparticles as solid phase carrier

60 pM

no/yes

51

upconversion nanoparticals based FRET

35 pM

yes (1% serum) /yes

52

TIRFM-based imaging method

6.5 aM

no/no

53

CONCLUSIONS In summary, we have developed an analytical platform based on optical tweezers with two-photon excitation for fluorescence detection of H5N1 gene sequences. The setup is cost-effective due to the use of a Q-switch nanosecond laser instead of expensive femtosecond pulse laser for TPE. Moreover, as a result of a heterogeneous enrichment method for DNA using microspheres and signal detection using an extremely sensitive detector (EMCCD), the detection limits for target DNA are 16 ~ 19 pM with no need for DNA amplification. Most importantly, due to the fact that TPE fluorescence emission is generated in the restricted three-dimensional vicinity of the trapping center (focal point), thus the excitation of background fluorescence is avoided and the assay is separation free,

this

approach can be expanded to straightforward detect target DNA in human whole serum with powerful anti-interference ability and have good selectivity for one-base mismatched, which laid a firm foundation for its future biological and clinical application, such as detection of tumor markers and virus in medical samples. To some extent, this promising platform with a simple procedure and high-throughput may open a new way for analytical sciences.

SUPPORTING INFORMATION AVAILABLE

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Supporting Information Available: supporting figures S1-S5 and additional experimental details of optical trapping and manipulation of a single hybrid-conjugated microsphere. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +86-27-68756759. Fax:+86-27-68754067. †

These authors contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21275110, 81572086, 21535005, 21275111) and the Fundamental Research Funds for the Central Universities (2042014kf0246). REFERENCES (1) Ashkin, A.; Dziedzic, J. M.; Yamane, T. Nature 1987, 330, 769-771. (2) Ashkin, A.; Dziedzic, J. M. Science 1987, 235, 1517-1520. (3) Mao, F. L.; Xing, Q. R.; Wang, K.; Lang, L. Y.; Wang, Z.; Chai, L.; Wang, Q. Y. Opt. Commun. 2005, 256, 358-363. (4) Zhong, M. C.; Wei, X. B.; Zhou, J. H.; Wang, Z. Q.; Li, Y. M. Nat. Commun. 2013, 4, 1768. (5) Harris, J.; McConnell, G. Opt. Express 2008, 16, 14036-14043. (6) Pang, Y.; J.Gordon, R. Nano Lett. 2012, 4, 402-406. (7) Sivaramakrishnan, S.; Sommese, R.; Sung, J. M.; Ali, M.; Doniach, S.; Flyvbjerg, H.; Spudich, J. A. Biophys. J. 2010, 98, 24-26. (8) Toshimitsu, M.; Matsumura, Y.; Shoji, T.; Kitamura, N.; Takase, M.; Murakoshi, K.; Yamauchi, H.; Ito, S.; Miyasaka, H.; Nobuhiro, A.; Mizumoto, Y.; Ishihara, H.; Tsuboi, Y. J. Phys. Chem. C 19



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Scheme 1. Schematic diagram of the principle for quantitative detection.

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Figure 1. Schematic drawing of the optical tweezers setup. The red line and the yellow line show the optical paths for 1064 nm laser for both optical trapping and TPE, and the LED lamp for bright-field illumination, respectively. The two-photon fluorescence signal (the orange line) is detected by a cooled EMCCD. The inset shows the details of the closed sample chamber with thickness of approximately 0.84 mm. NDF: neutral density filter; M1, M2: mirrors; L1-L4: lens; DM: dichroic mirror; SPF: short-pass filter.

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F

Figure 2. (A) The square relationships between the fluorescence intensity and the mean laser power on sample for a trapped microsphere tagged with 605 nm QDs with 100 ms exposure time. (B) The square relationships between the fluorescence intensity and the mean laser power on sample for a trapped microsphere tagged with 705 nm QDs with 150 ms exposure time. (C) TPE Fluorescence stability of 605 nm and 705 nm QDs conjugated PS microspheres.

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Figure 3. Quantitative detection of NA genes in buffer solution. (A) The linear relationship between the fluorescence intensity and the concentration of T-NA in the range of 0.5 to 30 nM with 100 ms exposure time. (B) The linear relationship between the fluorescence intensity and the concentration of T-NA in the range of 0 to 300 pM with 800 ms exposure time. (C) The corresponding real time images of T-NA in the range of 30 to 0.5 nM. (D) The corresponding real time images of T-NA in the range of 300 pM to 0 pM. Scale bar is 3 µm for all cases.

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Figure 4. Fluorescence images of the hybrid-conjugated microspheres in the presences of 30 nM target DNA and labeled with 605 nm QDs (A) and 705 nm QDs (B), respectively, which were recorded by an inverted fluorescence microscope under green light excitation with 60 ms exposure time. The scale bars for both fluorescence images are 6 µm.

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Figure 5. Quantitative detection of HA genes in buffer solution. (A) The linear relationship between the fluorescence intensity and the concentration of T-HA in the range of 0.5 to 30 nM with 150 ms exposure time. (B) The linear relationship between the fluorescence intensity and the concentration of T-HA in the range of 0 to 300 pM with 1000 ms exposure time. (C) The corresponding real time images of T-HA in the range of 30 to 0.5 nM. (D) The corresponding real time images of T-HA in the range of 300 pM to 0 pM. Scale bar is 3 µm for all cases.

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Figure 6. TPE fluorescence images of hybrid-conjugated microspheres enriching 30 nM NA genes in buffer solution (A) and whole serum (B), respectively. Fluorescence images of hybrid-conjugated microspheres enriched with 30 nM HA genes in buffer solution (C) and whole serum (D), respectively. All the images were recorded by a digital camera with 3s exposure time and the scale bar is 6 µm. (E) The fluorescence intensity of completely complementary sequences, one-base mismatched sequences, two-base mismatched sequences and random sequences for NA genes and HA genes, respectively.

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SYNOPSIS TOC

An analytical platform based on optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences was proposed. The approach was further expanded to directly detect target DNA in human whole serum without any separation steps. The detection limit was as low as 16 ~ 19 pM with good selectivity for one-base mismatched DNA sequences.

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Fluorescence Detection of H5N1 Virus Gene Sequences Based on

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