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Multiplexed Detection of Attomole Nucleic Acids Using Fluorescent Nanoparticle Counting Platform Xiaojing Pei, Haoyan Yin, Tiancheng Lai, Jun-Long Zhang, Feng Liu, Xiao Xu, and Na Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04551 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017
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Analytical Chemistry
Multiplexed Detection of Attomole Nucleic Acids Using Fluorescent Nanoparticle Counting Platform Xiaojing Pei,† Haoyan Yin,§ Tiancheng Lai,† Junlong Zhang,§ Feng Liu,† and Xiao Xu*,‡, 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, P. R. China § Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and Applications, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China ‡ Division of Nano Metrology and Materials Measurement, National Institute of Metrology, Beijing, 100029, P. R. China Correspondence should be addressed to Dr. Na LI. Tel: +8610 62761187; email:
[email protected]. Correspondence may also be addressed to Dr. Xiao XU. Tel: +86 10 64526538; email:
[email protected].
ABSTRACT: The sensitive multiplexed detection of nucleic acids in a single sample by a simple manner is of pivotal importance for diagnosis and therapy of human diseases. Herein, we constructed an automatic fluorescent nanoparticle (FNP) counting platform with a common fluorescence microscopic imaging setup for non-amplification multiplexed detection of attomole nucleic acids. Taking the advantages of the highly bright, multi-color emitting FNPs and magnetic separation, the platform enables the sensitive multiplexed detection without the need for extra fluorescent labels. Quantification for multiplex DNAs, multiplex microRNAs (miRNA), as well as the DNA and miRNA mixture was achieved with the similar dynamic range, the limit of detection down to 5 amol (5-µL detection volume), and the 81–115% spike recovery from different biological sample matrices. In particular, the sensitivity for multiplex miRNA is by far amongst the highest without using amplification or the lock nucleic acid hybridization enhancement strategy. Results about miRNA-141 from four different cell lines were agreeable with the quantitative reverse transcription polymerase chain reaction. Simultaneous detection of miRNA-141 and miRNA-21 in four different cell lines yielded consistent results with publications, indicating the potential for monitoring multiplex miRNA expression associated with the collaboratively regulation of important cellular events. This work expands the rule set of multiplex nucleic acid detection strategies and shows promising potential application in clinical diagnosis.
To be able to detect multiple nucleic acid sequences in a single sample is undoubtedly highly beneficial and vastly demanded for diagnosis and therapy of human diseases.1-4 However, the simple, sensitive and cost-effective multiplex detection is extremely challenging, and the available approaches are limited.5-7 To this end, much efforts have been made to improve the sensitivity by using amplification strategies or sensitive detection modes, such as the enzymatic and non-enzymatic target or signal amplifications,8-20 the plasmonic enhanced detection,21-27 the nanopore sensor,28-31 and the signal quenching-recover strategy,32-36 to name a few. However, these approaches have either limited multiplexing or quantification capabilities, and some need sophisticated setup and implementation.37,38 It is clear that more efforts are still urgently needed to develop simple and sensitive methods for the multiplexed detection. By analyzing all possible routes to the multiplexed detection, it can be seen that the signal readout mode is a central element that imparts influences on the method sensitivity, the multiplexing capacity and the instrumentation cost.8,39,40 Based on the characterized frequency/wavelength of the optical labels, the spectroscopic signal readout, such as surface enhanced Raman scattering (SERS) and fluorescence spectroscopy, can easily realize the multiplexed detection. However, spectrum-based quantification is often problematic due to the ensemble intensity measurements.41 In SERS, the intensity reproducibility for quantification can be compromised due to the variation of the enhancing substrate, and
instrumentation is still costly for routine analyses.42-44 In fluorescence measurements, the spectral overlap and the inner-filtering quenching effect amongst fluorophores inevitably jeopardize the accuracy and capacity in multiplexed quantification.45,46 The array technology can markedly improve the multiplexed detection capability of fluorescence measurements.47-49 However, an extra label is needed for most quantification, complicating encoding/decoding procedures.12,50 The fluorescent microsphere suspension arrays may also suffer from the ensemble intensity measurements.2,51,52 The microwell array imaging based counting is a type of highly sensitive signal readout and can circumvent the problem from ensemble intensity measurements by producing a digital signal,50 thus, holds a great promise in multiplex nucleic acid detection. However, the sophisticated setup and instrumental cost are often unaffordable for resource-limited settings.53,54 Therefore, it is imperative to further develop simple, sensitive, and cost-effective signal readout platforms for multiplex nucleic acid detection. We herein for the first time constructed a fluorescent nanoparticle counting platform with microscopic imaging for the multiplexed detection of attomole nucleic acids. Combined with the magnetic separation, fluorescent nanoparticles (FNPs) associated with the target of interest are used for both coding and quantitative purposes. We first demonstrated the advantages of the proposed platform in sensitivity and sample volume requirements by comparing with spectrofluorometry and flow cytometry. We then
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evaluated the figures of merit for the quantitative detection of multiplex DNAs, microRNAs (miRNAs), as well as the DNA and miRNA mixture. Finally, we assessed the potential of the established platform for practical applications by measuring the spike recovery of multiple nucleic acids from biological sample matrices. Particularly, we corroborated the reliability and accuracy of the proposed counting platform by comparing miRNA-141 (miR141) from four cell lines with the quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also simultaneously detected miR-141 and miR-21 in four different cell lines to demonstrate the potential of the proposed counting platform for monitoring multiplex miRNA expression associated with the collaboratively regulation of important cellular events. This work paved the way for practical applications in non-amplification detection of multiplex nucleic acids. EXPERIMENTAL SECTION Materials. All synthetic DNA oligonucleotides were purchased from Sangon Biotech Co., Ltd (Shanghai, China). All HPLCpurified miRNAs and RNase inhibitor were purchased from Takara Biotechnology Co., Ltd (Dalian, China). 1-Ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC, 99%), auric chloride dehydrate (A.R.), and Triton X100 (C.P.) were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). Na2HPO4, NaCl, NaH2PO4, NaOH, all of A. R. grade, were obtained from Beijing Chemical Works (China). 2-(NMorpholino) ethanesulfonic acid (MES) was obtained from J&K Chemical Ltd (Beijing, China). DRM-700 CELL-VU® CBC hemacytometers were obtained from Advanced Meditech International, Inc (New York, USA). Diethylpyrocarbonate (DEPC)treated water was purchased from VWR International Co., Ltd (Pennsylvania, USA). The miRcute miRNA isolation kit DP501 was purchased from Tiangen Biotech Co., Ltd (Beijing, China). SYBR Mix (Cat. No. 04913914001) was purchased from La Roche Ltd (Basel, Switzerland). M-MLV Reverse Transcriptase (Cat. No. 28025013) was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Wahaha® purified water was used throughout for all DNA study. The RNase-free environment was created throughout the experiments by using DEPC-treated water and RNase-free tips and tubes. Fluorescent nanoparticles, FC02F/10308 with size of 200 nm, 10865 with size of 220 nm, and 11242 with size of 250 nm, were purchased from Bangs laboratories, Inc (Indiana, USA). Streptavidin-modified magnetic beads (DynabeadsTM MyOneTM Streptavidin T1, d =1 µm, 10 mg/mL) were purchased from Thermo Fisher Scientific (Massachusetts, USA). Quantitative assay of the single target. The quantitative assay of targets was carried out according to the procedure as follows. And the same design and procedure are applicable to miRNAs. In a typical experiment, the detection of DNA was carried out in PBS containing 0.1% Triton X100. The assay mixture with a total volume of 100 µL containing 10 µL of DNA-modified FNPs (29 fM green FNPs, 58 fM red FNPs, 80 fM blue FNPs for specific assay), 40 µL of 2 mg/mL DNA-MBs and 10 µL of target DNA with concentration as indicated (10− 10000 pM). Specifically, 40 µL of 2 mg/mL DNA-MBs, 40 µL of PBS containing 0.1% Triton X100, and 10 µL of target DNA were added to the Eppendorf tube. The mixture was cooled from 75 °C to 37 °C with slow agitation, and then kept at 37 °C for 60 min to allow the hybridization between target molecules and capture probes on the MB surface. Afterward, 10 µL of DNA-modified FNPs (29 fM green FNPs, 58 fM red FNPs, 80 fM blue FNPs for specific assay) was added, and the mixture was cooled from 37 °C to 25 °C with slow agitation and then kept at 25 °C for 120 min to allow formation of
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the sandwich structure between DNA-FNP, target, and DNA-MB. After magnetic separation, the supernatant was removed and the assemblies were washed five times with PBS containing 0.1% Triton X100. Finally, 100 µL of 0.15 M NaOH was added to dehybridize and release FNPs from the sandwich composite. After 15 min, 5 µL of the supernatant was applied on the hemacytometers and the image was obtained with the fluorescence microscope. Quantitative assay of multiplex targets. For the duplex target assay, the final assay mixture had a total volume of 100 µL, containing 20 µL of 4 mg/mL DNA-MBs for each target sequence, 10 µL of DNA-modified FNPs (29 fM green FNPs, 58 fM red FNPs, 80 fM blue FNPs for different group), and 10 µL of each target sequences with concentrations as indicated (20−10000 pM), respectively. The same procedure as for the singleplex DNA detection was followed. For triplex target detection, the final assay mixture had a total volume of 100 µL, containing 10 µL of 29 fM green FNPs, 10 µL of 58 fM red FNPs and 10 µL of 80 fM blue FNPs and 10 µL of 8 mg/mL DNA-MBs for each target sequence, and 10 µL of each target sequence with concentrations as indicated (20−10000 pM), respectively. The same procedure as for the singleplex DNA detection was followed. The spike recovery of nucleic acids from cell lysate. The cervical cancer cell lines (HeLa) are incubated in complete medium (Dulbecco’s Modified Eagle’s Medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin) at 37 °C in atmosphere containing 5% CO2. Incubated HeLa cells were collected by trypsinization and centrifugation, washed with PBS, and pelleted at 3000 rpm for 5 min at 4 °C. The cells were re-suspended in PBS containing 0.1% Triton X100 at a concentration of about 106 cells/mL, incubated for 30 min at −20 °C, and then centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was collected and filtered through a 0.45 µm filter membrane prior to storing at −20 °C. For singleplex target detection, 10 µL of target and 20 µL of the cell lysate were added to the assay mixture to make a final assay volume of 100 µL; for singleplex miRNA detection, 10 µL of the cell lysate was added; for multiplex nucleic acid detection, 5 µL of the cell lysate was used. Assays without the standard targets were carried out in parallel. The quantification was carried out by following the same procedure as described above, and the spike recovery was calculated. The spike recovery of miRNA from total RNA extract. The spike recovery of miRNA, including miR-21, let-7c, and miR-141 were detected in total RNA extract from about 106 HeLa cells. The same cell incubated procedures as described above. The total RNA in the cells was extracted using the miRNA isolation kit from Tiangen Biotech (Cat.No. DP501) according to the manufacturer's instruction. The purified total RNAs were eluted with RNase-free water and the final volume was 30 µL. Ten microliters of target miRNA with concentrations as indicated and 10 µL of extracted total RNA were added in the assay mixture with the final volume of 100 µL. Assays without the standard targets were carried out in parallel. Then, quantification was carried by following the same procedure described above, and the spike recovery was calculated. Fluorescence microscopic imaging of FNPs. All fluorescence microscopic images were acquired using the “pixel shifting” mode of the Olympus CellSense software with a resolution of 1360 × 1024 pixels. The 10× objective lens was used for singleplexed assay and the 20× objective lens was used for multiplexed assay.
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Analytical Chemistry
Scheme 1. Schematic illustration of the assay rationale and the procedure for nucleic acid detection based FNP imaging and counting. In this study, 200-nm FNPs were used for producing signal readout. The number of nanoparticles was acquired through color recognition of the dots in the images based on the color characteristics. Images were acquired with Olympus IX73 under the following imaging condition: for green nanoparticles (λex/em: 480/520 nm), exciter 472 nm, emitter 520 nm and dichroic 495 nm; for the blue nanoparticles (λex/em: 360/450 nm), wide band UV excitation, exciter filter with excitation band 340 nm-390 nm, dichroic beams flitter with dichroic mirror at 410 nm, barrier emission filter 420 nm; for red nanoparticles (λex/em: 525/600 nm), exciter 562 nm, emitter 641 nm, dichroic mirror at 593 nm. The imaging condition was kept consistent during the experimental process of each reaction system unless indicated. For singleplexed assay, each counting result was the average of six images; for multiplexed assay, each counting result was the average of eight images. Color image processing. The 3 colors of FNPs were recognized and enumerated automatically using the software developed in C# programming language based on our previous work.55,56 The general idea of the automatic counting was to recognize the FNPs referred sequentially by shape and color characteristics. Specifically, at first, Gaussian blur was applied to smooth the image, and, sharpen edges was used to enhance the edge of the FNPs. The high-pass filtering step described in our previous works was then applied to eliminate off-focus FNPs signals interfering with the recognition. The shape-based segmentation was then used to divide the image into subimages with each containing a single object to be identified. The shape (area and axial ratio) and color judgments were sequentially applied to each subimage to identify FNPs, and last the number of FNPs was counted. The three colors of FNPs were found to be best separated in the
CIELCh color space, with C* (chroma) and h° (hue angle) components. To simplify the identification, an average color was generated for each subimage. The average color had color differences (calculated with CIEDE2000 algorithm57) less than a specified threshold with more than a half of the pixels of the object in a subimage. Average colors obtained from the images of each single type of FNPs were considered as reference colors. Linear boundaries of the reference color in C*- h° chart were then calculated and used as the criteria for color judgments. RESULTS AND DISCUSSION Scheme 1 illustrates the assay workflow and the color-based FNP counting with fluorescence imaging for the multiplexed detection of nucleic acids. Specifically, red, green and blue emitting fluorescent polystyrene nanoparticles (~200 nm) are assigned to three different target sequences. Recognition of the target, DNA or miRNA, is based on the formation of the typical sandwichstructured nanocomplex amongst the target, the capture DNAfunctionalized magnetic bead (DNA-MB), and the capture DNAfunctionalized FNP (DNA-FNP). DNA-FNPs are then released after the magnetic separation, and imaged with the fluorescence microscope. Subsequently, the automatic color-based particle identification and counting are implemented to provide the quantitative information based on counts of FNPs. The FNP counting is achieved via the recognition of the colored dots in the images based on the color characteristics. In the CIELCh color space, FNPs can be characterized with C* (chroma) and h° (hue angle) components. Specifically, criteria boundaries were (C*>10, 40°
