Nonamplification Sandwich Assay Platform for Sensitive Nucleic Acid

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Letter

A non-amplification sandwich assay platform for sensitive nucleic acid detection based on AuNPs enumeration with the dark-field microscope Tian Li, Xiao Xu, Guoqing Zhang, Ruoyun Lin, Yang Chen, Chenxi Li, Feng Liu, and Na Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00535 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on March 31, 2016

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Analytical Chemistry

A non-amplification sandwich assay platform for sensitive nucleic acid detection based on AuNPs enumeration with the dark-field microscope Tian Li,† Xiao Xu,‡ Guoqing Zhang,§ Ruoyun Lin,† Yang Chen,† Chenxi Li,† Feng Liu,† Na Li*,† †

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China ‡

Division of Nano Metrology and Materials Measurement, National Institute of Metrology, Beijing, 100029, China

§

Suzhou Nanomicro Technology Company Limited, Suzhou, 215123, China

ABSTRACT: A simple and efficient assay platform with high sensitivity, convenient implementation, and moderate cost in reagents and instrumentation is most appropriate for routine applications. Based on the gold nanoparticle (AuNP) enumeration signal readout mode established in our laboratory, we have developed a non-amplification sandwich assay for nucleic acid detection with the 3 fM limit of detection for a sequence related to the Alzheimer’s disease. This AuNP counting based method takes advantages of the distinctive and strong localized surface plasmon resonance light scattering with the dark-field microscope and magnetic separation. It is shown that the presence of 20-nM random DNA sequence or calf thymus DNA with the mass up to 106 fold of the targets do not significantly interfere with the assay signal. The spike recoveries of Hela cell lysate sample at 109.3% for 20 pM target and 110.5% for 100 pM target indicate the potential of this proposed method in practical sample applications. This nonamplification sandwich assay platform in principle is applicable to other assays such as the immunoassay, thus would be expected to find a breadth of applications that can make the best use of the simplicity and sensitivity.

The ability for rapid and sensitive detection of nucleic acids becomes vitally important in a variety of fields such as food safety, environmental monitoring and clinical diagnostics.1-3 These practical needs have driven the development of a breadth of novel ultrasensitive detection methods, including optical and electrochemical measurements combined with the enzymatic or non-enzymatic amplification strategies.4-14 However, apart from the high sensitivity, practical applications require one or more additional features, such as simple and easy implementation which involves minimum number of experimental steps, low cost chemical reagents as well as instrumentation, portability for field detection, and multiple target detection.2, 15 Among the different strategies, gold nanoparticles-based assays meet most of these requirements and have drawn considerable research interest.4, 6, 16-20 Gold nanoparticles (AuNPs) can be easily and reproducibly prepared,21 and the easy surface modification of gold surface diversified the functionalization options.16 Furthermore, the attractive optical and electronic attributes can be fine-tuned by the size, shape, inter-particle distance, as well as surface properties associated with the creation of chemical and biological sensors.22-24 Particularly, the featured localized surface plasmon resonance (LSPR) endows AuNPs with distinctive color and strong intensity at the LSPR wavelength.25-28 According to the Mie theory, the

plasmonic cross-section produces a 10- to 10,000-times higher signal than fluorescent dyes or quantum dots,29 thus gold nanoparticles (d>40 nm) can be easily detected with the simple dark-field microscope (DFM) with lower-cost, white light illumination.30-32 As a simple, sensitive and cost-effective technique, the dark-field microscope provides a powerful tool to the direct image AuNPs at the single nanoparticle level.22, 33 Therefore, dark-field imaging based biosensing has gained increasing attention in bioanalytical, diagnostic and therapeutic applications.24, 34-38 In dark-field imaging based methods, the spectral shift and the scattered intensity32, 35, 37, 39-42 as well as the number of particles43-46 can all be used for quantitative purposes. However, the available spectral change based approaches generally suffer from expensive instrumentation due to the requirement for tracking single particles and measuring the spectrum, and particularly quantification of the target based on spectral shift from limited number of single particles is generally not realistic for practical applications.32, 41-42 Furthermore, the spectrum is tightly associated with the size and morphology of nanoparticles, thus the imperfection of nanoparticle monodispersity and morphology often compromises the detection sensitivity and reproducibility. Particle counting method is a strategy correlating the number of AuNPs with the amount of the target molecules

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Scheme 1 Schematic illustration of the assay rationale of the proposed method. The DNA sequences used in this study are provided in Table S1.

which are invisible under the dark-field microscope. As the requirement for high-end instrumentation and nanoparticle monodispersity is markedly reduced, the particle-counting approach has the great potential for practical applications. For the particle-counting approach, it is imperatively important that the counting method can effectively reduce, if not eliminate, interferences from the sample matrix or microscope slide surface. We in our previous work developed an automatic counting method for the quantification of gold nanomaterials by applying the high-pass filtering operation and introducing color criteria to minimize the aforementioned interferences.46 To promote this simple AuNPs enumerating detection mode in biosensing applications, we in this work aimed to develop a simple, cost-effective and sensitive assay method for DNA detection without involving the amplification step. Scheme 1 illustrates the rationale of the proposed method which combines the sandwich-type DNA hybridization strategy and the magnetic separation. The DNAfunctionalized AuNP (AuNP-DNA) serves as the probe for capturing the target DNA as well as the transducer to produce the detectable signal with the dark-field microscope. In the presence of target DNA molecules, the AuNP-DNA probe and biotin-modified DNA capture probe (probebiotin) can be brought into proximity by forming a sandwich structure (AuNP-DNA, target and probe-biotin), which can be collected and separated from excess AuNPs using streptavidin-modified magnetic beads. The targetbound AuNPs are then released and separated from the magnetic beads via de-hybridization. Subsequently, released AuNPs are applied onto the amine-modified coverslip, on which the negatively charged AuNPs can be evenly distributed and stabilized to facilitate high quality image acquisition. Since the level of target DNA is highly correlated to the number of AuNPs, by counting the number of AuNPs in the dark-field image, the amount of target DNA can thus be calibrated and determined (Figure 1). In this work we chose a sequence region from human chromosome 19 as the target to demonstrate the applicability of this automatic counting assay. Recent research revealed

that the A/G single-nucleotide variation (rs4147918) in this sequence showed significant association with the Alzheimer’s disease.47

Figure 1. Dark-field images of AuNP-DNA with different concentrations of 40-nucleotide target DNA.

In order to optimize the assay procedure, we evaluated major factors that might affect the analytical performance. The number of DNA capture strand attached to a gold nanoparticle can influence both the stability of AuNPs and the hybridization efficiency. In the functionalization of AuNPs with the capture DNA, DNA with varied concentrations (0.5, 1.0 and 2.0 µM), was used to modify AuNPs (41 pM) to adjust the density of DNA on the gold nanoparticle surface. For 100-pM target DNA, the best signal-tobackground ratio was achieved when 1 µM DNA was used to modify AuNPs (Figure S5), which produced approximately 326 (standard deviation was 14) strands on one 59nm particle (Figure S4). The amount of AuNP-DNA is an important factor that exerts influence on the analytical performance of the assay. It is basically required that the amount of capture DNA modified AuNPs is high enough to avoid multi-target binding of a single AuNP, thus, the amount of target can be quantitatively correlated with the amount of AuNPs finally counted. However, the sensitivity


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can be compromised at the high AuNP level, because the non-specific binding between magnetic beads and AuNPs may increase the background signal. For 100-pM target DNA, the best signal-to-background ratio was achieved with 31-pM AuNPs (Figure S6). It would be desirable that a DNA detection method can be applied to DNA target with varied lengths to offer more adaptability to diversified length of sequences. Our proposed method was found to be suitable for targets with the length of single-stranded DNA from 24 to 60 nucleotides. Under optimized experimental conditions, the target DNA with concentrations ranging from 20 pM to 20 nM was able to be quantified with the limit of detection (by the 3σ/k rule, n=6) at 3 fM, 0.6 pM and 13 pM for 24, 40 and 60 nucleotides, respectively (Figure 2). Taking the 40nucleotide target DNA as an example, the AuNPs counting method showed higher sensitivity than the colorimetric method as well as the naked-eye detection. With naked eyes or extinction spectra, the detectable DNA target concentration was 100 pM (Figure S7 and Figure 2d). The transmission electron microscopic images from the supernatant also presented the increased number of AuNPs with the increased concentration of the target (Figure S8). Single-stranded DNA often forms stable secondary structures under assay conditions.48 Therefore, as the sequence becomes longer, the region of interest may be involved in intramolecular hybridization to hinder the hybridization with capture probes. This complication may result in compromised sensitivity. Nevertheless, to the best of our knowledge, the sensitivity of the proposed simple and costeffective method is the highest amongst the currently published non-amplified DNA sensing approaches based on the LSPR property of AuNPs, including dark-field microscopic imaging, light scattering and colorimetry.7, 17, 32, 41, 44 Particularly, our method avoided the demand for complicated reagents or many thermal cycles with precise control of temperature, thus is a simple approach that may facilitate practical uses.

Figure 2. Automatic counting results for target DNA with different lengths, (a) 24 nucleotides, (b) 40 nucleotides and (c) 60 nucleotides; (d) the photograph of the supernatant with different concentrations of 40-nucleotide target DNA (from left to right, 0 pM, 20 pM, 100 pM, 500 pM, 4 nM and 20 nM).

In addition to the sensitivity, the selectivity is also an important figure of merit for the performance of the propose method. We first evaluated the detection result of the target DNA in the presence of a random sequence of 42nucleotide DNA (Figure 3a). AuNP counts did not response to the concentration of the random sequence, suggesting that the random sequence did not exert interfering effect on the signal. With the existence of 20-nM random sequence, a linear response to the concentration of target DNA was still obtained with the limit of detection of 7.1 pM. We further evaluated the detection method in the presence of calf thymus DNA (ctDNA) which was used as an imitator for a complicated matrix (Figure 3b). No significant change in the counting results was observed with the target DNA/genomic DNA ratio (in mass) up to 1:106. This result indicated that interference from the genomic DNA was minimal and the nonspecific absorption was not significant in the test range.

Figure 3. (a) AuNP counts as a function of the concentration of a random sequence and the 40-nucleotide target DNA in the presence of 20-nM random sequence. Inset: the logarithm plot of AuNP counts versus target DNA concentration. The random sequenced DNA is 5’-GGGTTGGGTGGGACTCACCAGCTCCA ACTACCACAAGTTTGG-3’. (b) AuNP counts with 100 pM (black bars) or 0 pM (gray bars) 40-nucleotide target DNA in the presence of increasing amounts of ctDNA. Dashed line is the mean values of results from the ctDNA only.

To test the potential of the proposed method for more practical applications, we further applied this method to



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detect target DNA in Hela cell lysate, because cell lysate is realistically complex matrixes containing a variety of proteins, genomic DNA, total RNA, and other contaminants. Specifically, a diluted Hela cell lysate sample was spiked with target DNA at two concentration levels. It was found that the percent recovery of the spike (n=3) was 109.3% for 20 pM target and 110.5% for 100 pM target with the relative standard deviation (RSD) of 9.2% and 4.7%, respectively. These preliminary results showed that the proposed method has the feasibility for application with biological samples. In summary, we have successfully demonstrated a nonamplification strategy for nucleic acid detection at the femtomolar level based on automatic AuNPs counting with the dark-field microscope. This method combines the highly sensitive single plasmonic nanoparticle counting technique with the magnetic separation to achieve a better analytical performance with a simple procedure. The particle counting approach avoids the attachment of the expensive spectrometer to the dark-field microscope setup and is immune to the heterogeneity of the nanoparticle size and morphology. Additionally, the method can be implemented without the need for involving enzyme and other complex reagents, as well as tedious procedures. Furthermore, this sandwich-binding design in principle makes this detection platform easily extendable to other targets of interest.

ASSOCIATED CONTENT Supporting Information The Supporting Information including details of characterization of gold nanomaterials and experimental details is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *Tel.: +86 10 62761187. Fax: +86 10 62751708. E-mail: lina@ pku.edu.cn.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21475004, 21275011, 21535006, and 21505125).

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Scheme 1 Schematic illustration of the assay rationale of the proposed method. The DNA sequences used in this study are provided in Table S1. 59x21mm (300 x 300 DPI)



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Nonamplification Sandwich Assay Platform for Sensitive Nucleic Acid

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