Fluorescent Biosensors Based on Single-Molecule Counting

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Fluorescent Biosensors Based on Single-Molecule Counting Fei Ma,†,§ Ying Li,‡,§ Bo Tang,*,† and Chun-yang Zhang*,† †

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China ‡ Medical School, Shenzhen University, Shenzhen 518060, China CONSPECTUS: Biosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors. Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo labeling of target biomolecules with proper fluorescent tags, the quantification of certain biomolecule at the single-molecule level is achieved. In comparison with conventional ensemble measurements, single-molecule detection-based analytical methods possess the advantages of ultrahigh sensitivity, good selectivity, rapid analysis time, and low sample consumption. Consequently, single-molecule detection may be potentially employed as an ideal analytical approach to quantify low-abundant biomolecules with rapidity and simplicity. In this Account, we will summarize our efforts for developing a series of ultrasensitive biosensors based on single-molecule counting. Single-molecule counting is a member of single-molecule detection technologies and may be used as a very simple and ultrasensitive method to quantify target molecules by simply counting the individual fluorescent bursts. In the fluorescent sensors, the signals of target biomolecules may be translated to the fluorescence signals by specific in vitro/in vivo fluorescent labeling, and consequently, the fluorescent molecules indicate the presence of target molecules. The resultant fluorescence signals may be simply counted by either microfluidic device-integrated confocal microscopy or total internal reflection fluorescencebased single-molecule imaging. We have developed a series of single-molecule counting-based biosensors which can be classified as separation-free and separation-assisted assays. As a proof-of-concept, we demonstrate the applications of single-molecule counting-based biosensors for sensitive detection of various target biomolecules such as DNAs, miRNAs, proteins, enzymes, and intact cells, which may function as the disease-related biomarkers. Moreover, we give a summary of future directions to expand the usability of single-molecule counting-based biosensors including (1) the development of more user-friendly and automated instruments, (2) the discovery of new fluorescent labels and labeling strategies, and (3) the introduction of new concepts for the design of novel biosensors. Due to their high sensitivity, good selectivity, rapidity, and simplicity, we believe that the singlemolecule counting-based fluorescent biosensors will indubitably find wide applications in biological research, clinical diagnostics, and drug discovery.

1. INTRODUCTION The progress in biological research and clinical practice is greatly dependent on the development of powerful methodologies for accurate quantification of biomolecules. For a long time, this issue is explored by conventional biochemical techniques such as gel electrophoresis, 1 enzyme-linked immunosorbent assay (ELISA),2 and colorimetric assay.3 Although these methods enable efficient quantification of biomolecules, the insufficient sensitivity and cumbersome procedures limit their practical applications, especially the measurement of low-abundant targets. For example, the concentrations of disease-related protein biomarkers in serum are usually in the range from 10−16 to 10−12 M, which cannot be © XXXX American Chemical Society

measured by conventional immunoassays whose detection limit is above 10−12 M.4 Therefore, great efforts should be put into the design of new biosensors with improved sensitivity and simplicity. Remarkably, single-molecule fluorescence detection approaches show promising potential in the development of ultrasensitive biosensors. Single-molecule fluorescence detection originated form the measurement of single dopant molecule in a p-terphenyl crystal at cryogenic temperatures in 1989 and 1990.5,6 Subsequently, the first image of single carbo Received: May 18, 2016

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Figure 1. Integration of FRET with single-molecule counting for DNA detection. (A) (a) The presence of target DNA leads to the formation of Cy5-target DNA-QD assembly. (b) Illumination of QD induces FRET from the QD to Cy5. (c) Experimental setup for single-molecule detection. (B) Fluorescent bursts of Cy5 in the absence (right) and in the presence (left) of target DNA. Adapted with permission from ref 24. Copyright 2005 Nature.

cyanine dye at room temperature was obtained with near-field scanning optical microscopy in 1993.7 Later, single-molecule imaging at room temperature was acquired with far-field microscopy in 1996.8 These important advances pave the way for its biological applications. Its first demonstration is the imaging of single fluorescently labeled myosin molecule and the measurement of individual ATP turnover reaction by total internal reflection fluorescence (TIRF) microscopy.9 Since then, single-molecule fluorescence detection has become a powerful analytical technique in the fields of physics, chemistry, and biology.10−12 Some typical examples include the study of heterogeneous diffusion in thin polymer films,13 the discovery of crystal-face-dependent TiO2 photocatalysis,14 and the revealing of sequence-specific misfolding in multidomain proteins.15 In contrast to the monitoring of ensemble average by conventional methods, single-molecule approaches enable the measurement of individual molecules. Although multiple fluorescence properties (e.g., emission wavelength, fluorescence decay time, and photon burst size) can be used for quantitative analysis,16 single-molecule fluorescence counting may be the simplest and most direct way for the quantification of target molecules.17 Once the target is fluorescently labeled, the fluorescent burst indicates the presence of target molecules, and the counting of fluorescent bursts is just the counting of target molecules. Some exciting works have been reported recently, such as the use of single-molecule counting for the quantification of proteins in a single cell,18 the measurement of chemical reaction of individual enzyme molecules,19 the detection of single protein in complex mixtures,20 and the quantification of miRNA expression.21

This Account will focus on our efforts in developing singlemolecule counting-based fluorescent biosensors. Two kinds of microscopy instrument (i.e., confocal microscopy and TIRF microscopy) are used in our experiments to obtained singlemolecule imaging. Confocal microscopy is the main far-field method for single-molecule imaging.22 In the confocal microscopy, a small pinhole (50−100 mm in diameter) is placed in the image plane to reject the light from the out-offocus regions, defining a focused volume of 0.5−1.0 femtoliters in the specimen.23 This small detection volume may effectively reduce the background noise. To ensure the accuracy, we integrated a microfluidic device with the confocal microscopy for single-molecule counting.24 Alternatively, TIRF microscopy is based on the total internal reflection phenomenon that occurs when light passes from a high-refractive medium (e.g., glass) into a low-refractive medium (e.g., cell and water).25 The evanescent field produced by total internally reflected light only excites the fluorescent molecules in a thin layer with approximately 100 nm thickness immediately next to the reflection interface, thus the background noise emanating from the inner depth is efficiently minimized. Both confocal and TIRF microscopies work well in a wide range of conditions including ambient atmosphere and biological environment, enabling them suitable for biosensor development. Single-molecule counting has significant advantage of ultrahigh sensitivity, high signal-to-noise ratio, low sample consumption, and rapid analysis time, making them an ideal platform for quantitative biosensing. Our single-molecule counting-based biosensors involve two main steps: fluorescent labeling and single-molecule counting. In some cases, singlemolecule imaging is required prior to the counting.26 Generally, our single-molecule counting-based biosensors may be divided B

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HIV-2 genes may be achieved. In comparison with the reported multiplex assay,39 this biosensor possess significant advantages of high sensitivity, short analysis time, and extremely low sample consumption.

into two categories: separation-free and separation-assisted assays.

2. SEPARATION-FREE SINGLE-MOLECULE COUNTING-BASDED BIOSENSORS Our separation-free biosensors are based on the Förster resonance energy transfer (FRET) technology.24,27,28 FRET is characterized by a distance-dependent and nonradioactive energy transfer from a high-energy donor fluorophore to a lower energy acceptor fluorophore. The occurrence of energy transfer requires a spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, a spatial proximity of the donor and acceptor, and a nonperpendicular orientation of the transition dipole moments.29 FRET techniques are very sensitive due to its extended apparent Stokes’ shift,30 and the target biomolecules may be measured in a homogeneous manner without the involvement of washing and separation steps. We integrated FRET with single-molecule counting, and developed a series of ultrasensitive biosensors with quantum dots (QDs) as the energy donor. In contrast to organic fluorophores and fluorescent proteins, the QDs have distinct advantages of good photostability, high quantum yield, sizetunable photoluminescence spectra with narrow emission bandwidth, making them ideal fluorescent labels for biosensing.31−34 The single-QD-based FRET biosensors have been successfully applied for sensitive detection of various biomolecules.

2.2. Detection of MicroRNA

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs responsible for gene expression regulation.40 The dysregulation of miRNAs is closely associated with various human diseases.41 Because of their small size, high sequence similarity, and low expression level, sensitive detection of miRNAs remains a great challenge. Northern blotting is the most prevalent method for miRNA assay,42 but it is usually laborious with large sample consumption. Real-time reverse transcription PCR provides an alternative way,43 but it involves complicated sample pretreatment. Since cellular miRNA concentration is as low as 1000 molecules per cell,44 sensitive detection method is highly desirable. We developed an isothermal amplification-assisted single QD-based biosensor for ultrasensitive detection of miRNA (Figure 2A).27 The introduction of a two-stage exponential amplification reaction may greatly amplify the target miRNA, and the resultant products may subsequently hybridize with a set of capture and reporter probes to form the sandwich hybrids, which may assemble on the 605QD surface to obtain the 605QD/reporter

2.1. Detection of DNA

The alteration in DNA sequence is closely associated with various human diseases including Alzheimer’s disease, sickle cell anemia and cancers.35 Conventional DNA assay often requires either preamplification or separation procedures to improve the sensitivity.36,37 To simplify the assay, we introduced FRET to develop a separation-free biosensor (Figure 1A).24 This biosensor consists of a streptavidin-conjugated 605QD, a biotin-modified capture probe and a Cy5-tagged reporter probe. The presence of target DNA leads to the formation of capture probe-DNA-reporter probe hybrids. Then the streptavidin-conjugated 605QD functions as both the energy donor and the concentrator to capture multiple sandwiched hybrids, bringing Cy5 and the 605QD into close proximity and consequently the occurrence of FRET from the QD to Cy5. The DNA concentration may be simply monitored by counting the Cy5 bursts. In comparison with the ensemble FRET assay, our single-QD-based biosensor has significant advantages of zero background fluorescence and improved FRET efficiency, enabling sensitive detection of low-abundant targets. Notably, ∼50 copies of DNA can be detected without preamplification. Moreover, this biosensor may be further applied for the discrimination of K-ras point mutations in clinical samples from patients with ovarian cancer. Single-QD-based biosensors may integrate FRET with coincidence detection for simultaneous detection of multiple DNA targets.38 The 605QD and two dyes (i.e., Alexa Fluor 488 and Alexa Fluor 647) were used in this assay. At the excitation wavelength of 488 nm, both the 605QD and Alexa Fluor 488 may be excited simultaneously, generating a coincidence signal. Meanwhile, Alexa Fluor 647 may emit fluorescence as a result of FRET from the 605QD to Alexa Fluor 647. Through simply counting the coincidence signals and Alexa Fluor 647 fluorescence bursts, simultaneous detection of HIV-1 and

Figure 2. (A) Integration of two-stage exponential amplification reaction with single-molecule counting for miRNA assay. (a−c) Generation of reporter oligonucleotide Y through two-stage exponential amplification reaction. (d) Construction of reporter probe/reporter oligonucleotide Y/capture probe sandwiched hybrid. (e) Assembly of sandwiched hybrids on the 605QD surface. (f) Emission of Cy5 induced by FRET from the 605QD to Cy5. (B) Change of Cy5 burst counts with miRNA concentration. Reprinted with permission from ref 27. Copyright 2012 American Chemical Society. C

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Accounts of Chemical Research oligonucleotide/Cy5 complexes. Upon excitation with a wavelength of 488 nm, distinct Cy5 signal can be detected as a result of FRET from the QD to Cy5. The Cy5 counts may be used to quantify the target miRNA with a detection limit of 0.1 aM (Figure 2B). This assay is 4 orders of magnitude more sensitive than the single-QD-based nanosensor without amplification.24 This biosensor can discriminate single-nucleotide differences between different miRNA family members of let-7a, let-7b, and let-7c. We further integrated primer generation-mediated rolling circle amplification (RCA) with FRET, and developed a single QD-based biosensor for miRNA point mutation assay.47 Recent research demonstrates that the increased expression of mir196a2C (T → C mutation in mir-196a2 miRNA) is closely associated with the shortened survival of non small-cell lung cancer (NSCLC) patients.46 In our assay, the miRNA point mutation of mir-196a2C is discriminated by specific padlock probes and subsequently amplified by primer generationmediated-RCA. The resultant amplified products may be simply monitored by single QD-based biosensor, and the target concentration can be accurately quantified by the measurement of Cy5 counts. This assay exhibits high sensitivity with a detection limit of 50.9 aM, which has improved by as much as 2 orders of magnitude as compared with the reported ensemble RCA assay.47 Moreover, this assay possesses a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM. Especially, this biosensor can be applied for mir-196a2 assay in lung tissues of NSCLC patients. 2.3. Detection of Enzyme Activity

Figure 3. Integration of FRET with single-molecule counting for the detection of renin (A), PKA (B), and telomerase (C) activities. (A) The presence of renin causes the cleavage of Cy5-labeled peptide substrate and the release of Cy5 from the QD surface, inducing the decrease of Cy5 emission. (B) PKA catalyzes the incorporation of γbiotin-ATP into the Cy5-labeled-substrate peptides and subsequently the assembly of Cy5 on the QD surface, inducing the increase of Cy5 emission. (C) The presence of telomerase induces the addition of Cy5-dATP to the primer by extension reaction, leading to the assembly of Cy5 on the QD surface and consequently the FRET from the QD to Cy5. Reprinted with permission from refs 48, 28, and57. Copyright 2012 American Chemical Society, 2015 American Chemical Society, and 2015 The Royal Society of Chemistry, respectively.

We demonstrate the development of a signal-off single-QDbased biosensor for renin assay.48 Renin plays important role in the regulation of blood pressure and electrolyte homeostasis,49 and the excessive secretion of renin may cause congestive heart failure and cancers.50 In our assay (Figure 3A), Cy5-labeled peptide substrate of renin is immobilized on the 605QD surface to form a 605QD/substrate/Cy5 complex, leading to the occurrence of FRET from the QD to Cy5. The presence of rennin may cause the cleavage of peptide substrate and induce the release of Cy5 form the QD surface and consequently the decrease of FRET efficiency. The renin activity may be quantified by the measurement of Cy5 counts. The sensitivity of this assay has improved by more than 40-fold than the reported ensemble FRET-based method.50 Moreover, this biosensor can accurately determine the enzymatic velocity and the Michaelis−Menten kinetic parameters of renin. We further demonstrate the development of a signal-on single-QD-based biosensor for cAMP-dependent protein kinase (PKA) assay.28 Protein kinases play crucial roles in intracellular signal transduction and metabolic pathways, and their alteration may cause various human diseases such as neurodegenerative diseases and cancers.51 Conventional detection methods include radioactive assay52 and immunoassay53 with the involvement of hazardous materials and complicated procedures. In our assay (Figure 3B), the PKA-catalyzed phosphorylation reaction specifically incorporates γ-biotinATP into the Cy5-labeled-substrate peptides to generate biotinylated peptides, which may assembles onto the 605QD surface to form a Cy5/peptide/QD complex through specific biotin−streptavidin interaction, generating an efficient FRET signal. The PKA activity can be quantified by the measurement of Cy5 counts. This assay exhibits high sensitivity with a detection limit of 9.3 × 10−6 U/μL, which is 10 times more

sensitive than the reported fluorescent method,54 and can be used for the screening of PKA inhibitors and activators. We also developed a signal-on single QD-based sensor for telomerase activity assay. Telomerase is a reverse transcriptase which may specifically add repeat units to the ends of a telomere. The up-regulation and reactivation of telomerase activity are found in over 85% of human cancers.55 Polymerase chain reaction (PCR)-based telomere repeat amplification protocol is one of the most general approaches for telomerase assay,56 but it is time-consuming and requires precise control of temperature cycling. In our assay (Figure 3C),57 the presence of telomerase induces the extension reaction to add Cy5-dATP to the 3′-end primer. The resultant Cy5-labeld primer may subsequently hybridize with the biotinylated capture probe to form a Cy5-labeled biotinylated dsDNA, which may assemble on the 605QD surfaces to obtain a Cy5-dsDNA-QD assembly, inducing FRET from the QD to Cy5. The telomerase activity can be quantitatively detected by monitoring the Cy5 counts. This assay is very simple without the involvement of any amplification step, and possesses high sensitivity with a detection limit of 7 cells/μL. It can be further applied to D

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CLIP-tag, and the SNAP/CLIP-tag can be covalently labeled by O6-benzylguanine (BG)/O2-benzylcytosine (BC) derivatives, respectively.62 Moreover, the BC/BG derivatives are conjugated with ATTO 488/Alex Fluor 647, respectively, to form BCATTO 488/BG-Alex Fluor 647. As a result, SUMOylation signals may be translated to distinguishable fluorescence signals and simply monitored by single-molecule counting. This biosensor can measure the SUMOylations under various physiological conditions. To overcome the steric hindrance of SNAP/CLIP tag (size of ∼20 kDa) to the SUMO chain formation, we employed the tetracysteine (TC) tag (size of 1) generates brighter fluorescence spots than single SUMO2 (n = 1), thus the protein polySUMOylation may be evaluated in a quantitative manner by measuring the number and the brightness of fluorescence signals. This biosensor can be used to measure Sp100 polySUMOylation under different physiological conditions.

screen telomerase inhibitors for the discovery of new anticancer drugs.

3. SEPARATION-ASSISTED BIOSENSORS Even though the integration of FRET with single-molecule counting frees from washing and separation steps, efficient FRET requires the careful consideration of the distance and the angle between the donor and the acceptor, limiting its practical applications. To simplify the assay design, we develop a series of separation-assisted biosensors based on single-molecule counting. These biosensors rely on efficient separation techniques (e.g., magnetic separation and affinity chromatography) to eliminate the background signal, and are suitable for multiplex detection of protein modifications, HIV genes, and histone modifying enzymes.26,58,59 3.1. Detection of Protein Modifications

The functions of proteins are usually regulated by various posttranslational modifications.60 SUMOylation, the attachment of small ubiquitin-like modifier (SUMO) chains to the target protein, is one of the most important protein post-translational modifications. SUMOylation is responsible for the regulation of various cellular processes and is closely associated with human diseases such as Parkinson’s diseases, type 1 diabetes, and cancers.61 Conventional SUMOylation assay includes computation-based SUMOylation predication and mass spectrometry with the limitation of only theoretical output and complicated data analysis. We integrated the SNAP/CLIP-tag labeling with single-molecule counting, and developed a simple approach for simultaneous measurement of intracellular SUMOylations (Figure 4A).26 In this research, we used p53 (a tumor suppressor) and RanGAP1 (a GTPase-activating protein) as the model targets. The SUMO is genetically fused with SNAP/

3.2. Detection of HIV Genes

To achieve ultrasensitive detection of DNA without target amplification, we introduced a liposome-QD (L/QD) complex (Figure 5A).59 We prepared the L/QD complexes which encapsulated hundreds of QDs, L/QD complex-tagged reporter

Figure 4. (A) Integration of SNAP/CLIP-tag-mediated translation with single-molecule counting for simultaneous detection of protein SUMOylation. The assay involves three steps: (a) simultaneous SUMOylation in vivo, (b) fluorescent labeling with SNAP/CLIP-tag, (c) TIRF imaging, and (d) single-molecule counting. (B) Combination of TC tag labeling with single-molecule counting for the measurement of protein polySUMOylation. The polymeric SUMO chains (n > 1) generate a brighter fluorescence signal than the single SUMO entity (n = 1). Reprinted with permission from refs 26 and 63. Copyrigt 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, and 2014 American Chemical Society, respectively.

Figure 5. (A) Integration of magnetic separation with single-molecule counting for simultaneous detection of multiple DNA targets. This assay involves (a) preparation of L/QD complexes, L/QD complextagged reporter probes and magnetic bead-modified capture probes, (b) formation of sandwich hybrids in the presence of target DNA and the subsequent magnetic separation, and (c) release of QDs from L/ QD complex and the measurement by single-particle counting. (B) Fluorescence bursts of the released QDs in the presence of HIV-1 (a), HIV-2 (b), and both HIV-1 and HIV-2 (c). Reprinted with permission from ref 59. Copyright 2013 American Chemical Society. E

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peptide substrates at H3K14 and H3K9 positions, respectively. The modified sites may be subsequently recognized by two dyetagged antibodies through antibody−antigen interaction, translating the information on two enzymes to distinguishable fluorescence signals. After magnetic separation, the released fluorescent molecules can be simply quantified by singlemolecule counting (Figure 6B). This biosensor exhibits excellent specificity and high sensitivity with a detection limit of 21 pM for GcN5 and 12 pM for histone G9a, respectively. The sensitivity for GcN5 assay has improved by 1 order of magnitude as compared with that of ensemble QD-based FRET assay,65 and the sensitivity for G9a assay is 1 order of magnitude higher than that of AuNP-based colorimetric assay.66 This biosensor can be further applied for the screening of HME inhibitors.

probes, and magnetic bead-modified capture probes, respectively. The presence of target DNA leads to the formation of L/ QD-DNA-hybrid-magnetic-bead assembly, which can be removed from the unbound reporter probes by magnetic separation. The disruption of L/QD complexes by chloroform releases the QDs which can be simply quantified by singleparticle counting (Figure 5B). Simultaneous detection of HIV-1 and HIV-2 genes may be easily achieved by using two sets of capture and reporter probes and two different-color L/QD complexes. The detection limit of this assay may reach 1 aM for HIV-1 and 2.5 aM for HIV-2, respectively, which has enhanced by as much as 3 orders of magnitude as compared with that of single QD-based biosensor.24 3.3. Detection of Histone-Modifying Enzymes

Histone modifying enzymes (HMEs) are responsible for catalyzing the addition/removal of various covalent modifications of histones including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP ribosylation. The disorders of HME activity may cause developmental defects and various diseases.64 We developed a simple method for multiplex detection of HMEs through the integration of antibody-based fluorescent labeling with single-molecule counting (Figure 6A).58 We used histone acetyltransferase GcN5 and histone methyltransferase G9a as the model enzymes, which can specifically modify the biotin-labeled

3.4. Detection of Cancer Cells

In addition to the detection of various biomolecules, singlemolecule counting-based biosensor may be used for direct detection of intact cells. Conventional cancer cell assay usually relies on the morphological criteria, but it is not suitable for the identification of cancer types.67 Recently, we developed a simple and sensitive method for multiplex detection of lung cancer cells at the single-molecule level (Figure 7A).67 We used

Figure 7. Integration of magnetic separation with single-molecule counting for multiplex detection of lung cancer cells. (A) Target cancer cells are recognized by biotinylated capture probes and fluorescent dye-tagged reporter probes, respectively. After magnetic separation, the released fluorescent molecules may be monitored by single-molecule counting. (B) Variance of the measured number of fluorescent molecules with the number of A549 (left) and H23 (right) cells. Reprinted with permission from ref 67. Copyright 2014 The Royal Society of Chemistry.

two biotinylated aptamers as the capture probes for the recognition of different cancer cells, two fluorescent dye-tagged aptamers as the reporter probes for the generation of distinct fluorescence signals. After magnetic separation, a single cancer cell may release hundreds of thousands of reporter probes, which can be simply quantified by single-molecule counting (Figure 7B). This biosensor can simultaneously discriminate two human lung adenocarcinoma cells (i.e., A549 cells and H23 cells). In comparison with the RCA-based chemiluminescence assay, the sensitivity of this assay has improved by more than 10

Figure 6. Integration of magnetic separation with single-molecule counting for multiplex detection of HMEs. (A) Information on HMEs (i.e., GcN5 and G9a) may be transferred to distinct fluorescence signals by antibody labeling. After magnetic separation, the released fluorescent molecules may be simply quantified by single-molecule counting. (B) Fluorescence signals of Alexa Fluor 488 (a−d) and Alexa Fluor 647 (e−h) in the absence of target (a, e) and in the presence of GcN5 (b, f), G9a (c, g), and both GcN5 and G9a (d, h). Adapted with permission from ref 58. Copyright 2016 The Royal Society of Chemistry. F

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University. His research focuses on analytical chemistry and nanotechnology. Chun-yang Zhang obtained his Ph.D. degree from Peking University, China, in 1999. During 1999−2008, he worked at Tsinghua University, China; Emory University, Atlanta, GA; The Johns Hopkins University, Baltimore, MD; and The City University of New York, New York. In 2009, he joined as a professor in Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, China. In 2015, he relocated to Shandong Normal University, China. He is the recipient of the China National Fund for Distinguished Young Scientists. His research focuses on analytical chemistry, bionanotechnology, and single-molecule detection.

4. CONCLUSIONS AND OUTLOOK Due to its significant advantages of ultrahigh sensitivity and low sample-consumption, single-molecule counting may serve as an ideal technology for the development of sensitive biosensors. In this Account, we briefly review our efforts in the development of a series of sensitive biosensors based on single-molecule counting. We have designed FRET-based separation-free biosensors and separation-assisted biosenors, and demonstrated their potential applications in the detection of DNAs, miRNAs, proteins, enzymes, and even intact cells.24,26−28,45,57,58,63,64,69 These biosensors are quite simple and sensitive, and they exhibit good performance even in complex biological samples, holding great potential for further application in clinical diagnostics and drug discovery. Despite the rosy prospects of single-molecule counting-based biosensors, great efforts should be put into expanding their usability. First, the instrument used in single-molecule detection is usually sophisticated and expensive, and the development of more user-friendly and automated instruments with affordable prices will undoubtedly make them become accessible to more interested persons.70 Second, taking into account the possible applications in harsh sample conditions, the discovery of new fluorescent labels and labeling strategies will be beneficial to keeping high and stable performance of biosensors under diverse situations. Third, the introduction of recent advanced concepts (e.g., aptamer71 and isothermal amplification72) may provide valuable direction for the development of novel biosensors with good selectivity and high sensitivity. We believe that the single-molecule counting-based biosensors will find wide applications in basic biomedical research and practically clinical diagnostics in the near future.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523, 21527811, and 31400655), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86 0531-86180010. Fax: +86 0531-86180017. E-mail: [email protected]. *Tel.: +86 0531-86186033. Fax: +86 0531-82615258. E-mail: [email protected]. Author Contributions §

F.M. and Y.L. contributed equally.

Notes

The authors declare no competing financial interest. Biographies Fei Ma obtained his MS degree from Nanjing Agricultural University in 2013. He worked at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences from 2013 to 2015. Currently, he is a lecturer at Shandong Normal University. His research focuses on biosensor development and single-molecule detection. Ying Li obtained her Ph.D. degree at Huazhong University of Science and Technology in 2009. She worked at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences from 2009 to 2015. Now she is a senior researcher at Medical School of Shenzhen University. Bo Tang obtained his Ph.D. degree at Nankai University in 1994. Since then, he has been working as a professor at Shandong Normal G

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DOI: 10.1021/acs.accounts.6b00237 Acc. Chem. Res. XXXX, XXX, XXX−XXX