Digital Concentration Readout of DNA by Absolute Quantification of

Oct 28, 2016 - (b) Changes in the total number of AuNRs confined in the enriched region with changes in the predicted number, shown in log–log space...
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Digital Concentration Readout of DNA by Absolute Quantification of Optically Countable Gold Nanorods Guohua Li,†,‡,§ Liang Zhu,†,‡,§ Zhenjie Wu,‡,§ Yonghong He,*,‡,§ Hui Tan,*,∥ and Shuqing Sun*,‡,§ ‡

Institute of Optical Imaging and Sensing, Shenzhen Key Laboratory for Minimal Invasive Medical Technologies, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, People’s Republic of China § Department of Physics, Tsinghua University, Beijing 100084, People’s Republic of China ∥ Shenzhen Key Laboratory of Neurosurgery, The First Affiliated Hospital of Shenzhen University, Shenzhen, 518035, China S Supporting Information *

ABSTRACT: Isolation and amplification procedures are indispensable for traditional single-molecule detection methods applied in low-abundance biomolecule analysis. Here, we describe a method for ultrasensitive detection of DNA through encoding a single target molecule with a single-countable nanometer-sized substitute (gold nanorod, AuNR) and enrichment of the substitute AuNRs into a limited region followed by accurate microscopic enumeration. The enrichment and the bright distinct color allow the AuNRs to be efficiently counted from as few as 1−2 to tens of thousands in 3 μL of test solution, which demonstrates the ability of rapid digital concentration readout of the target DNA. On this basis, a detection limit of 6.5 aM was achieved for DNA associated with human papillomavirus (HPV). Notably, our method requires neither complicated isolation and amplification procedures nor extremely expensive instruments and consumables.

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arrays of femtoliter-sized reaction chambers coupled with microscopic beads to isolate and detect single target molecules14−18 or the approach that converts an individual target molecule to a single-countable fluorescent micrometersized DNA molecule via rolling-cycle-amplification,19,20 and so on. However, among these methods, complicated and specialized facilities are required to isolate the individual target molecules. For example, stringent control of the enzymatic process to avoid its inherent instability and variability is required to amplify the single-molecule signal, and highly sensitive instruments are required to realize single-molecule detection.10,21−23 All these greatly increase the complexity of the experimental procedures and the costs, greatly limiting their applications on a broad scale. Therefore, a new simple and costeffective SMD method without the involvement of isolation and amplification procedures is desired. Herein, we report a new method for highly sensitive detection of DNA based on a combination of magnetic replacement procedures to convert the single target into a single-countable nanoparticle and an electrostatic adsorptionbased enrichment method to efficiently count the nanoparticles. As shown in Scheme 1, a target DNA molecule is sandwiched between a magnetic bead (MB) and a gold nanorod (AuNR)

ighly sensitive nucleic acid assays provide a wealth of information in the practice of biological research and diagnostic medicine, especially for early-phase diagnosis.1−3 Single-molecule detection (SMD), which is capable of unmasking the heterogeneous behavior of subpopulations and the target molecule trajectory hidden by the ensemble measurement,4 is the most sensitive technique in biochemistry analysis. In principle, if the test sample contains enough target molecules above the Poisson noise limit and all the targets are queried, this method can yield the highest accuracy in concentration quantification.5−7 Typically, the observation volume for single-molecule detection is in the range from subfemtoliter to picoliter to ensure a sufficiently high signal-tonoise ratio.8,9 Thus, it would take an extremely long time to interrogate several tens of microliters of sample solution. This significant limitation in detection time renders SMD difficult to perform for low-abundance detection. To solve this problem, researchers are currently focused on the divide-and-conquer approaches that dramatically increase the observation volume without sacrificing the ability to identify single molecules. Generally speaking, an individual target molecule in a test sample is first confined into tens of hundreds of compartments or more, and the single-molecule signal in an individual compartment is significantly amplified to make it easier to determine its presence. The most typical example is commercially available digital polymerase chain reaction (PCR).10−13 Others may include methods that make use of © XXXX American Chemical Society

Received: July 15, 2016 Accepted: October 19, 2016

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DOI: 10.1021/acs.analchem.6b02712 Anal. Chem. XXXX, XXX, XXX−XXX

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into a 20 mL round-bottomed flask with vigorous mixing for 1.5 min at room temperature. The color of this mixture changed to yellow-brown immediately, indicating the formation of seed solution for AuNR growth. Second, to grow AuNRs, 28 μL of seed solution, 20 mL of 200 mM CTAB solution, 400 μL of 24 mM HAuCl4, 50 μL of 40 mM AgNO3 solution, 160 μL of 100 mM ascorbic acid, and 150 μL of 5 M HCl were mixed thoroughly. The mixture was left standing at room temperature for several hours until the color turned blue, indicating the formation of AuNRs. The resulting solution was centrifuged for 10 min at 8500 rpm, and the pelleted beads were redispersed into 10 mL of 0.01 M CTAB. Modification of Gold Nanorods with Thiol-DNA. The protocol for loading thiol-DNA [5′-SH-(CH2)6-A AAA AAA AAA ACA GAA AAT GCT AGT GCT TAT-3′] onto AuNRs was mainly carried out according to previously reported work with some modifications.25 In this work, CTAB molecules capping the AuNRs were exchanged with a mixture of PVP and sodium dodecyl sulfate (SDS), which can be easily displaced by thiol-functionalized molecules such as thiol-DNA. AuNRs were suspended in 0.01 M phosphate-buffered saline (PBS), pH 8.0, and centrifuged for 10 min at 8500 rpm twice to remove excess CTAB. The resulting AuNRs were resuspended in 10 mL of 0.01 M PBS containing 0.3% SDS. The obtained AuNR suspension and a 10% (w/v) PVP solution in ethanol were mixed well in a round-bottom flask. This mixture was stirred overnight at 40 °C. After stirring, CTAB capping the AuNRs was gradually exchanged by the mixture of PVP and SDS. The mixture was centrifuged for 10 min at 8500 rpm and resuspended in 0.01 M PBS containing 0.03% SDS. This process was repeated three times, and the final pellet was resuspended in 1 mL of PBS. A portion (200 μL) of 3 mM TCEP-deprotected thiol-DNA solution in PBS buffer was added to the postexchanged AuNR suspension. This AuNR suspension was sonicated for a few seconds and left standing for 20 h. Salting solution (120 μL) containing 0.01 M PBS, 0.3 M NaCl, 4 mM MgCl2, and 0.03% SDS was added. This salting process was repeated 10 times at intervals of 0.5 h. After the last salting solution addition, the solution was left at room temperature for another 12 h. The salting-processed AuNRs were centrifuged for 10 min at 8500 rpm and resuspended in 0.01 M PBS several times to remove the unconjugated thiolDNA. The resulting AuNRs were stored in 200 μL of 1× Tris− ethylenediaminetetraacetic acid (EDTA) (TE buffer). Conjugation of Amino-DNA onto Magnetic Beads To Prepare Magnetic Bead Probes. The basic process to conjugate the capture probes onto MBs was carried out mainly according to the manufacturer’s instructions. EDC is used to form an amide bond between the primary amino group of the DNA and the carboxylic acid group on the MB’s surface. A sample (100 μL) of 20 mg/mL MB solution was washed with 50 mM MES buffer, pH 5.0, containing 0.03% SDS. After this was washed twice, 100 μL of 50 mM MES buffer was added and mixed. An aliquot (70 μL) of 100 μM amino-DNA (5′-ATA GAG AAT GTA TAT CTA TGG AAA AAA AAA A-NH2-3′) in MES buffer was added and mixed by vortexing, and then 10 μL of 50 mg/mL freshly prepared EDC in ice-cold MES buffer was added and mixed well by vortexing. The solution was incubated for 30 min at room temperature with slow tilt rotation. Three additional repetitions of adding EDC and incubation processes were required for more amino-DNA conjugation. MBs were collected by application of a magnet, and the supernatant was pipetted out for quantification of

Scheme 1. Schematic Illustration for Detection of Target DNA

by a two-step DNA hybridization process. Upon magnetic separation, free AuNRs are thoroughly removed. Following the affinity-based dehybridization procedures, the coding AuNRs in the sandwich complexes are dissociated from the surface of the MBs. In this procedure, a large excess of MBs and AuNR probes are applied to ensure that each target is encoded with a single AuNR and most of the coding AuNRs can be efficiently captured by MBs. In this way, the detection of DNA is therefore transferred to the counting of the coding AuNRs because every single target DNA is substituted by a single AuNR. The number of coding AuNRs is positively correlated with the number of target DNA molecules in solution. Subsequently, a positively charged glass slide is used to immobilize and confine the negatively charged coding AuNRs to a limited region through an electrostatic interaction, which allows absolute quantification of the coding AuNRs by use of a dark-field microscope on a practical time scale.

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EXPERIMENTAL SECTION Materials. Magnetic beads functionalized with carboxylic acid groups (230 nm in diameter) were purchased from Enriching Biotechnology Ltd. (3-Aminopropyl)triethoxysilane (APTES) was purchased from Nanjing Chen Gong Organic Silicon Material Co., Ltd. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was purchased from Tokyo Kasei Kogyo Co., and solutions were made just before use. Tris(2-carboxyethyl)phosphine (TCEP), hydrogen tetrachloroaurate(III) hydrate (HAuCl 4 ·3H 2 O), 2-(Nmorpholino)ethanesulfonic acid (MES), sodium borohydride (NaBH4), silver nitrate (AgNO3), cetyltrimethylammonium bromide (CTAB), poly(vinylpyrrolidone) (PVP, MW 8000), and ethanolamine were purchased from Sigma−Aldrich. All DNA sequences were purchased from the Beijing Genomics Institution. Synthesis of Gold Nanorods. The synthesis of AuNRs was carried out by a previously reported seed-mediated method.24 First, the seed solution was prepared by addition of 24 μL of 100 mM freshly prepared, ice-cold NaBH4 solution, 40 μL of 24 mM HAuCl4, and 4 mL of 100 mM CTAB solution B

DOI: 10.1021/acs.analchem.6b02712 Anal. Chem. XXXX, XXX, XXX−XXX

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Automatic Identification of Gold Nanorods. In addition to the red spots, a very small number of light spots originating from the inevitable defects of glass slides or impurities in solution are also visible in dark-field images.26−28 Carefully cleaning all the reaction vials is of major importance to reduce the interference from contaminant particles, but it is far from enough, especially in low-concentration detection. To solve this problem, a custom-coded batch-to-batch image recognition program based on Matlab was applied to exclude the disturbance from contaminant particles, remove manual artifacts, reduce the labor cost, and automatically count all the AuNRs. In this program, a region segmentation algorithm was first applied to identify the individual particles, containing AuNRs and contaminant particles, on the basis of a given light intensity threshold. Subsequently, the pixel area that each particle occupies is considered. Particles with larger or smaller pixel areas are removed from the images. The true colors of the rest of the particles with proper pixel area values are analyzed and plotted in International Commission on Illumination (CIE) color space, a commonly used powerful tool for quantitative description of the true color or spectral information on objects.29−31 The synthesized AuNRs in our method appear red in the dark-field microscope because their maximal localized surface plasmon resonance (LSPR) peaks are in the red part of the visible spectrum. This indicates that AuNRs are mainly distributed in the red region of CIE color space. On the basis of current experimental conditions, a quantifiable color-related gating was set to balance the need for positive identification of the AuNRs and the need to exclude contaminant particles.

conjugated amino-DNA. The average number of amino-DNA molecules conjugated onto an individual MB was calculated as ∼1.1 × 104 on the basis of the change in UV/vis absorbance at 260 nm before and after the conjugation process. The coated MBs were incubated in 50 μL of 50 mM ethanolamine in PBS, pH 8.0, for 60 min at room temperature with slow tilt rotation to quench the unreacted activated carboxylic acid groups on the MBs. The MBs were collected and washed four times with 100 μL of 50 mM Tris buffer to remove excess amino-DNA. The washed MBs were preserved in 1 mL in 1× TE buffer for storage. Fabrication of (3-Aminopropyl)triethoxysilane-Modified Slides. Glass slides were immersed in piranha solution (30% H2O2/98% H2SO4 = 3:7) for 6 h to remove organic materials and then sonicated in ultrapure water several times to remove dust as thoroughly as possible. Clean slides were dried in a vacuum drying oven at 60 °C for 3 h. With this treatment, an overwhelming majority of potential impurities that may have interfered with AuNR signals were removed, and large amounts of hydroxyl groups were successfully modified onto the surface of glass slides to achieve the following APTES grafting. Dried glass slides were incubated in 30% (v/v) APTES ethanol solution for 12 h and then extensively rinsed in ethanol with sonication four times. Prior to use, these APTES-modified slides must be dried in the vacuum drying oven at 60 °C. Detailed Detection Protocol. In a typical experiment, a total of ∼6 × 108 MB probes were added to a 2 mL microcentrifuge tube. An aliquot (200 μL) of the target DNA (5′-CCA TAG ATA TAC ATT CTC TAT TAT CCA CAC CTG CAT TTG CTG CAT AAG CAC TAG CAT TTT CTG T-3′) test sample solution containing 0.01 M PBS buffer, 0.01% SDS, and 0.15 M NaCl was added. With slow tilt rotation at 37 °C for 30 min, the hybridization reaction between target molecules and capture probes on the MB surface was conducted. A magnet was introduced to separate the MBs. After the washing procedure, the MBs were resuspended in 50 μL of solution containing 0.01 M PBS, 0.15 M NaCl, and 0.1% SDS. Subsequently, 5 μL of a 0.176 nM AuNR code solution was added. AuNR codes were hybridized with target molecules that bound to the MBs with slow tilt rotation at 37 °C for 1 h. A magnet was used to separate the MBs. The supernatant was removed and the MBs were washed with washing buffer (0.01 M PBS, 0.15 M NaCl, and 0.1% SDS) five times to remove all unreacted AuNRs. After the last washing procedure, the aggregated MBs were resuspended in 10 μL of ultrapure water and subsequently transferred into a clean 200 μL tube. The tube was placed in a 60 °C water bath for 10 min to dehybridize the double-stranded DNA. MBs were assembled to the bottom of the tube by a strong magnet. A smple (3 μL) of the supernatant was dropped onto the clean and dried APTESmodified slide. After the slide was allowed to stand for 10 min, a clean and dried coverslip was placed on the slide to prepare the microscope specimen for dark-field imaging. Dark-field Microscopic Imaging of Gold Nanorods. An upright optical microscope (BX53, Olympus) equipped with a numerical aperture (NA) = 1.20−1.43 oil immersion dark-field condenser was used to image the AuNRs. The white light source used here was a 150 W halogen lamp. A 40× objective was used to collect scattered light from the AuNRs. A color charge-coupled device (CCD) camera (DP73, Olympus, 1600 × 1200 pixels) with an integration time of 500 ms was used to capture AuNR images. The practical size of an obtained image was approximately 0.34 × 0.26 mm2.

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RESULTS AND DISCUSSION

As shown in Figure 1a, CTAB-capped AuNRs, with average length 44 nm and average width 21 nm, were synthesized and used as coding nanoparticles. The longitudinal LSPR peak of the AuNRs was located at 613 nm, as observed in UV−vis extinction spectra (Figure 1b). Replacement of CTAB with

Figure 1. (a) Typical TEM image of as-synthesized AuNRs. (b) Normalized extinction spectra of AuNRs before and after thiolated DNA conjugation. (c) Scheme of dark-field imaging of AuNRs. (d) Typical dark-field microscopic image of AuNRs. C

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distribution in CIE color space. We can see that most of the spots dotted in CIE color space are concentrated in a fixed region, while a tiny proportion is out of this region. Note that, to obtain this distribution, the sample used was ultrapure water exposed to the atmosphere for half an hour. Reasonably, particles located in this dense distribution are viewed as contaminant particles and are removed from the image in our image recognition procedure. Figure 3 shows the representative chromaticity distribution of AuNR solution in CIE color space. Large quantities of

PVP and conjugation of thiolated DNA probes onto the AuNR caused a red shift of 4 nm of the longitudinal LSPR band (Figure 1b), in agreement with prior reports.25 After introduction of PVP and thiol-DNA, the spectrum red-shifted, indicating a change in surface chemistry of the AuNRs. Surface potential measurements also confirm this conclusion. The CTAB-capped AuNR has a ζ potential of 46.5 mV because of the positively charged CTAB coating bilayer. In contrast, a negative ζ potential of −39.5 mV is observed after the DNA conjugation process, indicating successful conjugation of DNA molecules to the AuNRs. AuNRs tens of nanometers in size have very high light absorption and scattering in visible light and do not show the blinking behavior that quantum dots exhibit.32,33 The excellent optical scattering property accounts for their wide use in bioimaging and biosensing.34−36 Remarkably, the strong Rayleigh scattering and distinct color feature enable an individual AuNR to be robustly and unambiguously resolved by a nonscanning dark-field microscope (Figure 1c,d).29,37,38 However, due to the difference in brightness of the features recorded in the image, the identification of a bright feature as a AuNR is largely subject to an individual’s experience. This may result in various results from the same experiment. Despite this problem, it is also tedious and time-consuming. Therefore, the introduction of a standard identification procedure is still required to eliminate artificial biases. CIE color space, a mathematic abstraction of the color feature, is utilized to give a unified and quantifiable criterion for us to distinguish true AuNRs from potential contaminant particles. The chromaticity coordinates of the identified particles in the dark-field images were calculated by eqs 1 and 2,39,40 where IR, IG and IB are the intensity values of the RGB values obtained by 8-bit color CCD camera:

Figure 3. Chromaticity distribution of contaminated AuNR solution (a) before and (b) after the automatic recognition program.

The color feature of the contaminant particles was studied initially. Figure 2 shows a typical dark-field image of contaminant particles and the representative chromaticity

particles are concentrated in the red region of CIE colorspace, which is totally different from the region that represents the contaminant particles, shown in Figure 2b. Considering this remarkable difference, light spots dotted in this region are all viewed as AuNRs and counted one by one in our program. Compared with the highly dense dots in the red region, there are many spots scattered throughout that region. It is certain that most of these spots are contaminant particles. In addition, some of them may be AuNRs because the synthesized AuNRs are not uniform; a certain proportion of them may be gold nanospheres and AuNRs with smaller aspect ratios with shorter longitudinal LSPR peak wavelengths. Figure 4 displays the typical performance of the image recognition program in accurately identifying AuNRs. In addition to a large number of red AuNRs, the original darkfield image has some strong light spots, darker spots, and a large area of diffraction rings in the right region, originating from contaminant particles. After processing with the image recognition program, most of these contaminant particles are

Figure 2. (a) Typical image of ultrapure water sample exposed to air. (b) Chromaticity distribution of contaminant particles, shown in CIE color space.

Figure 4. Automatic recognition of AuNRs. (a) Original image of AuNRs. (b) AuNR image processed by the home-coded image recognition program.

⎛ 0.49 ⎛X⎞ 0.31 0.20 ⎞⎛ IR ⎞ 1 ⎜ ⎟⎜ ⎟ ⎜ ⎟ I ⎜ Y ⎟ = 0.17697 ⎜⎜ 0.17697 0.81240 0.01063⎟⎟⎜ G ⎟ ⎜ ⎝Z⎠ ⎝ 0 0.01 0.99 ⎠⎝ IB ⎟⎠

(1)

⎛X⎞ ⎛x⎞ 1 ⎜ ⎟ ⎜ y⎟ = ⎜Y ⎟ ⎜ ⎟ X Y Z + + ⎝z⎠ ⎝Z⎠

(2)

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enriched region can be completed in less than 3 min. Figure 5b shows that the number of AuNRs detected is in good agreement with the method based on measurements of AuNR size and gold elemental content.43 It also proves that AuNRs are individually dispersed in aqueous solution to achieve minimal aggregation. Even if there are only 1−2 AuNRs in a 3 μL sample, which is equivalent to a molar concentration of 0.5−1 aM, they can still be detected, due to the extremely high (nearly 100%) enrichment efficiency in lower concentrations. This approach combines dark-field microscopy and enrichment of AuNRs, exhibiting an increase of at least 4−5 orders of magnitude in sensitivity over methods based on high-sensitivity flow cytometry or dynamic light scattering,44,45 for which the gold nanoparticle concentrations of 5−20 fM or higher are required. The changing trend of the detected variance coefficient is largely consistent with the rule of Poisson statistics (Figure 5c), where the standard deviation is equal to the square root of the mean value of the counts.46 This indicates that the quantitative accuracy is dominated by Poisson statistics. Notably, test solutions in which the total counts range from 100 to 300, with intervals of as few as 50, can be clearly distinguished from each other (Figure 5d). To confirm the feasibility of utilizing AuNRs as signal probes to encode DNA hybridization, a mock DNA target sequence (5′-ACA GAA AAT GCT AGT GCT TAT AAA AAA AAA AAA AAA AAA AAA AAA A-3′) was sandwiched by the dT25 MB (Dynal Invitrogen Corp.) and an AuNR conjugated with complementary DNA sequences. The success of the two-step hybridization procedure was qualitatively evaluated by the ability to form core−satellite assemblies. As shown in Figure 6, hardly an AuNR could be observed on a MB surface in the control experiment where no complementary target is present. However, in the presence of target DNA with concentrations of 1 nM and 100 nM, large quantities of AuNRs are immobilized on the MBs to form the core (MB)−satellite (AuNR) like assemblies. The average number of AuNRs immobilized onto a MB was estimated to be ∼600 and 7000, positively correlated with the target concentration. The nonlinearity between the target concentration and the number of attached AuNRs is mainly due to the limited availability of AuNR codes, since the concentration of added AuNRs is less than 100 nM. A target oligonucleotide associated with human papillomavirus (HPV) was chosen as a model system to demonstrate the applicability of the current assay for highly sensitive DNA detection. The HPV-associated DNA is first sandwiched by MBs and coding AuNRs through DNA hybridization, and the released coding AuNRs are subjected to dark-field microscopic imaging and absolute enumeration after enrichment on the slide surface. Figure 7 shows the plot of the number of coding AuNR as a function of the target concentration. The obtained plot has a nearly linear dose curve within the concentration range. A limit of detection (LOD) as low as ∼6.5 aM is achieved by extrapolating the concentration at the background plus 3 times the standard deviation. For comparison, the detection sensitivity of the current approach has improved by ∼20-fold compared with digital enzyme-linked immunosorbent assay (ELISA)14 and by nearly 3 orders of magnitude compared with the method based on the single quantum dot (QD) nanosensor.47 The ability to achieve such high detection sensitivity in this assay derives from the highly accurate counting of coding AuNRs and the efficient coding of the target molecules.

successfully removed. Meanwhile, most of the AuNR still remain there, indicating the excellent applicability of our program. Even though the identification of a single AuNR can be achieved, the quantification of AuNRs at low concentrations (such as