Graphene oxide-circular aptamer based colorimetric protein detection on bioactive paper

Xue Lia, Xin Hea, Qiang Zhangb, Yangyang Changa and Meng Liu*a
aSchool of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, Dalian University of Technology, Dalian, 116024, China. E-mail: mliu@dlut.edu.cn; liumeng.525@163.com
bSchool of Bioengineering, Dalian University of Technology, Dalian, 116024, China

Received 21st May 2019 , Accepted 12th July 2019

First published on 12th July 2019


Paper-based sensor technology represents a new class of point-of-care (POC) diagnostic devices that is affordable, portable, rapid and scalable for manufacturing. One of the goals of developing new paper-based POC diagnostics is the need to recognize targets with high specificity and sensitivity. Herein, a graphene oxide-circular aptamer based assay is described for highly specific and sensitive detection of a target protein on bioactive paper. The circular aptamer was self-assembled onto graphene oxide to generate the biorecognition moieties, followed by the desorption reaction induced by the specific binding of the target. The released circular aptamer then hybridizes to paper-bound DNA primers, thus initiating the rolling circle amplification reaction to produce a long DNA molecule containing multiple horseradish peroxidase-mimicking DNAzyme units, which further catalyze the oxidation of substrates by H2O2 in the presence of hemin to yield a distinct colorimetric signal on paper. Under the optimal conditions, this dual amplification method can achieve detection of platelet-derived growth factor (PDGF) at a concentration as low as 100 pM. Overall, this strategy of paper-based nanobiosensors may hold great potential for rapid, accurate and inexpensive biomarker detection in POC applications.


Introduction

There is currently a significant need for the development of rapid and inexpensive point-of-care (POC) diagnostic tests to help improve global public health.1–3 Among various new technologies, paper-based POC diagnostics that are affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to the end user in low-resource settings (ASSURED) have attracted considerable interest in recent years.4–8 Such technology is already having an important impact on low-cost diagnostics,9 and several microscale paper-based analytical devices (μPADs) have already been demonstrated for a range of key analytes (e.g. ATP, glucose, liver proteins, blood typing),10–14 as well as other analytes.15–18

One of the key requirements for expanding the utility of μPADs is the need to integrate various biological reactions into the devices, including molecular amplification technologies that can improve the sensitivity of such devices for detection of low concentration targets. However, to date, μPADs mainly utilize simple antigen–antibody interactions15,19–22 or nucleic acid hybridization reactions,23–26 which restrict the potential of such devices for applications that require multi-step reactions to enhance sensitivity or produce robust readouts.

Nucleic acid molecules that possess a defined function (such as binding and/or catalysis) have found growing use as molecular recognition elements for diagnostic applications.27 Functional nucleic acids (FNAs) typically include DNA and RNA aptamers, ribozymes (RNA based enzymes) and deoxyribozymes (DNAzymes).28 When compared to more typical biorecognition elements (e.g., antibodies and enzymes), FNAs possess numerous advantages including their high stability, tunable binding affinity and specificity, in vitro production, ease of chemical modification and immobilization, and compatibility with standard nucleic acid amplification methods.29 Although various FNAs are available for a broad range of targets, and new FNAs for desired targets can be relatively easily isolated from random-sequence pools, FNAs have rarely been successfully exploited for the design of functional bioactive paper-based sensors for use as POC devices.14,30,31

In this study, we report on a paper-based assay that integrates highly stable circular aptamers as biorecognition elements with an efficient isothermal enzymatic DNA replication, rolling circle amplification (RCA),32–35 for colorimetric protein detection, which provides important advantages for POC diagnostic devices that can be used for sensitive detection of important clinical analytes in resource-limited settings.

Materials and methods

Oligonucleotides and other materials

All DNA oligonucleotides (ESI, Table S1) were obtained from Integrated DNA Technologies (IDT), and purified by standard 10% denaturing (8 M urea) polyacrylamide gel electrophoresis (dPAGE). T4 polynucleotide kinase (PNK), T4 DNA ligase and phi29 DNA polymerase (ϕ29DP) were purchased from MBI Fermentas (Burlington, Canada). γ-[32P]ATP and γ-[32P]dGTP were purchased from PerkinElmer. Human thrombin was obtained from Haematologic Technologies Inc. (Essex Jct., VT). All other chemicals were purchased from Sigma-Aldrich (Oakville, Canada) and used without further purification. Nitrocellulose membranes (HF 180) were purchased from Millipore. Graphene oxide (GO) was synthesized according to our previously reported method.36

Instrumentation

The autoradiograms images of gels were obtained using a Typhoon 9200 variable mode imager (GE Healthcare) and analyzed using Image Quant software (Molecular Dynamics). Paper well plates were printed using a Xerox ColorQube 8570N solid wax ink printer to generate a 96-well outline, and then heated at 120 °C for 2 min to melt the wax through the paper to form a hydrophobic barrier.37

Preparation of circular aptamer for PDGF (CApt1)

The circular aptamer was synthesized from 5′-phosphorylated linear DNA oligonucleotides through template-assisted ligation using T4 DNA ligase. A total of 200 pmol of LApt1 (or LApt2) was first mixed with 10 U PNK and 2 mM ATP in 50 μL of 1× PNK buffer A (50 mM Tris–HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine). The mixture was incubated at 37 °C for 30 min, followed by heating at 90 °C for 5 min. A volume of 250 pmol of CD1 (or CD2) was then added and heated at 90 °C for 5 min. After cooling to room temperature (RT) the mixture was left for 10 min, after which 20 μL of 10× T4 DNA ligase buffer (400 mM Tris–HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP, pH 7.8 at 25 °C) and 10 U T4 DNA ligase were added. The resulting mixture (total volume of 200 μL) was incubated at RT for 2 h before heating at 90 °C for 5 min to deactivate the ligase. The ligated circular aptamers were concentrated by standard ethanol precipitation and purified by dPAGE.

Preparation of bioactive paper

Streptavidin was first conjugated to the biotinylated template primers (TP) according to our previously reported method.37 Briefly, 1 nmol of TP1 (or TP2, see ESI for sequences) and 30 μL of streptavidin (2 mg mL−1) were added to 400 μL of PBS buffer (25 mM containing 150 mM NaCl, 5 mM MgCl2, pH 7.6). After incubating at RT for 2 h, free TP1 was removed by centrifugation through a 30 kDa molecular weight cut-off membrane (NANOSEP OMEGA, Pall Incorporation) at 5000g for 10 min. The resulting conjugates were washed twice with PBS buffer and collected. A desired volume of the above solution was then printed onto each test zone using a Scienion SciFlexArrayer S5 Non-Contact Microarray Printer and allowed to dry at room temperature. To reduce the nonspecific binding, the bioactive paper with printed DNA was blocked by immersion into PBS buffer containing 3% casein for 40 min. After washing, the paper sensor was dried. The mixtures of all-RCA reagents containing 10 U of ϕ29DP, 1 μL of 10 mM dNTPs, 2 μL of 100 μM hemin and 5 μL of 10% (w/v) pullulan were then printed on the zone. The bioactive paper was dried and stored in a desiccant container until used.

Radioactive assay of CApt1 release from the graphene oxide surface by PDGF

Radioactive CAP1 was prepared by labeling at the 5′ terminus with γ-[32P]ATP using T4 polynucleotide kinase according to the manufacturer's protocol, and purified by 10% dPAGE. The CApt1 (10 μM) was denatured by heating at 90 °C for 10 min and then immediately cooled on ice for 5 min. After incubation in the target-binding buffer (TBB, 50 mM Tris–HCl containing 100 mM NaCl, 1 mM MgCl2 and 0.02% Tween-20, pH 7.4) at RT for 20 min, CApt1 was gradually refolded. For the reaction in Fig. 2a, 200 nM CApt1 was first incubated with 2, 4, 6, or 8 μg mL−1 of GO in TBB (50 μL) at RT for 60 min. Each mixture was then centrifuged at 14[thin space (1/6-em)]000 rpm for 5 min at 4 °C. The obtained precipitate (P) and supernatant (S) that contained CApt1 were analyzed by 10% dPAGE. For the reactions in Fig. 2b, 200 nM.

CApt1 was incubated with 8 μg mL−1 of GO for 1, 5, 10, 30 or 60 min prior to centrifugation and further dPAGE analysis. For the reaction in Fig. 2e, 200 nM CApt1 was first incubated with 8 μg mL−1 of GO for 60 min at RT, then 100 nM PDGF was added to initiate the release reaction for different times before the centrifugation and dPAGE analysis. These samples were first heated at 90 °C for 5 min in 1× urea PAGE loading buffer before loaded onto the gel well.

RCA reaction in solution and on paper

200 nM of CApt1 was first incubated with 8 μg mL−1 of GO in 50 μL of TBB for 60 min at RT. Subsequently, 1 μL of PDGF stock solution with different concentrations was added. Following 20 min incubation, each mixture was centrifuged at 14[thin space (1/6-em)]000 rpm for 5 min at 4 °C. For the RCA reaction in solution, 10 μL of the obtained supernatant was transferred into a tube. 2 μL of 10× RCA reaction buffer (330 mM Tris acetate, 100 mM magnesium acetate, 660 mM potassium acetate, 1% (v/v) Tween-20, 10 mM DTT, pH 7.9), 5 U ϕ29DP, 1 μL of TP1 (20 μM) and 1 μL of dNTPs (10 mM) were introduced to the above mixture (total volume: 20 μL). The reaction mixture was incubated at 30 °C for 30 min before heating at 90 °C for 5 min. The resultant RCA products were analyzed by 0.6% agarose gel electrophoresis.

For the RCA reaction on paper, 10 μL of the above supernatant was pipetted into each test zone of the bioactive paper. Then 1 μL of 10× RCA reaction buffer was added. The reaction was allowed to proceed at room temperature for 30 min. For the radioactive assay, 0.5 μL of [α-32P]dGTP was added before the RCA reaction. The bioactive paper was then washed by immersion into PBS buffer and dried under nitrogen flow before scanning. The resulting images were analyzed using ImageJ software.

Stability of circular aptamer and linear aptamer in cell lysate

Cell lysate from the MCF-7 breast cancer cell line was prepared according to our previous method.36 A total of 100 nM of γ-[32P]-labeled CApt1 or LApt1 was incubated with 20 μL of freshly prepared cell lysate (106 cells) at 30 °C for 1 h, 3 h or 6 h. Afterward, the mixtures were heated to 90 °C for 5 min and analyzed by dPAGE.

Results and discussion

The working principle of the assay is illustrated in Scheme 1. The sensing system integrates a circular aptamer-target binding reaction in solution and RCA reaction on paper. Transmission electron microscopy image revealed that micrometer-sized GO was synthesized. Initially, the circular aptamer was expected to easily adsorb onto the GO surface by virtue of the non-covalent π–π stacking interactions and hydrophobic interactions.36,38–40 In this case, no free aptamers were present in the supernatant after centrifugation, thus making them unavailable for the RCA reaction on paper. It is well-known that the graphene-adsorbed aptamer can undergo conformation changes upon target binding.36 Thus it was anticipated that the adsorbed circular aptamer could be released from the GO surface upon the addition of its cognate target, allowing the RCA reaction to be switched “on” on paper. As a result, the sensing of a target protein could be easily converted to the detection of RCA products (RP), thus enabling high-sensitivity protein detection.
image file: c9ay01060b-s1.tif
Scheme 1 Schematic illustration of the graphene oxide-circular aptamer based assay on bioactive paper.

To verify the feasibility of the proposed design, we set out to engineer a sensing platform for platelet-derived growth factor (PDGF), an important protein related to tumor growth and cell transformation.41–44 A circular aptamer (named CApt1) was first constructed that contained two key parts: (1) a well-characterized aptamer domain known to bind PDGF; (2) a primer binding domain that was required for the RCA reaction. Circular aptamers were chosen for this work owing to their superior stability relative to linear aptamers. This was demonstrated by challenging both the linear and circular aptamers with cell lysates (Fig. S1). As shown in Fig. 1, increasing amounts of LApt1 were digested by nucleases within the lysate with increasing incubation time, as can be deduced from the reduced intensity of radioactive bands for the intact LApt1 on dPAGE. As much as 92% of LApt1 was digested following a 3 h incubation time. In direct contrast, only 14% of CApt1 was digested after exposure to the same cell lysate for 3 h, confirming the high stability of circular aptamers against cellular nucleases.


image file: c9ay01060b-f1.tif
Fig. 1 Comparison of the stability of circular aptamer and linear aptamer against degradation by nucleases in cell lysates.

We next examined the adsorption of CApt1 onto GO. To achieve the complete adsorption, 200 nM of CApt1 was premixed with different amounts of GO for 1 h, followed by dPAGE analysis to determine the percentage of aptamer in the supernatant (S) and the precipitate (P) after simple centrifugation. As shown in Fig. 2a, CApt1 was observed exclusively in the precipitant due to the formation of a self-assembled CApt1-GO hybrid. When the concentration of GO was 8 μg mL−1, up to 99% of CApt1 was found in the P fraction. The kinetics of aptamer binding to the GO were also monitored. The result, shown in Fig. 2b, indicated that the adsorption was completed in 1 h.


image file: c9ay01060b-f2.tif
Fig. 2 Analysis of the adsorption and desorption of CApt1 on GO. (a) Adsorption of CApt1 on GO with different GO concentrations. (b) Time-dependent adsorption of CApt1 on GO. CApt1 (200 nM) was incubated with GO (8 μg mL−1) for 1, 5, 10, 30, 60 min prior to centrifugation and analysis. (c) Desorption of adsorbed CApt1 and CApt1M induced by PDGF. CApt1 or CApt1M (200 nM) and GO (8 μg mL−1) were first incubated for 1 h, followed by incubation with PDGF (100 nM) for 2 h prior to centrifugation and dPAGE analysis. (d) Desorption of adsorbed CApt1 induced by PDGF and non-target proteins. CApt1 (200 nM) and GO (8 μg mL−1) were first incubated for 1 h, followed by incubation with PDGF, BSA, thrombin, IgG, and cell lysate for 2 h prior to centrifugation and dPAGE analysis. (e) Kinetic analysis of desorption of CApt1 from GO. CApt1 (200 nM) and GO (8 μg mL−1) were first incubated for 1 h, followed by incubation with PDGF (100 nM) for different times prior to centrifugation and dPAGE analysis.

For the GO-adsorbed CApt1, the addition of PDGF (100 nM) resulted in the release of 56 ± 5% of CAPt1 into the supernatant (Fig. 2c), suggesting that the protein caused the displacement reaction. To confirm that the desorption process was a result of specific binding between PDGF and CApt1, we carried out two control experiments. First, a circular aptamer with mutations (CApt1M) was used to replace CApt1. It was expected that the desorption reaction would be inhibited since some nucleotides crucial for target binding were mutated in CApt1M. The addition of PDGF had little effect on the release of the adsorbed CApt1M. In the second control, several non-targeted proteins (BSA, thrombin, IgG) and a complex matrix (cell lysate) were used instead of PDGF. No significant signal was observed in the presence of any of these non-target species (Fig. 2d). Taken together, the displacement reaction was highly dependent on the correct target. To quantitatively study the release, the kinetics of desorption were monitored. As shown in Fig. 2e, the amount of released CApt1 increased with increasing the incubation time.

We next examined the RCA reaction in solution. In the absence of GO, CApt1 was able to function as a normal circular template for the DNA replication process both with (Fig. 3, lane 2) and without PDGF (lane 4), based on the appearance of the expected characteristic RP. Note that RP with large size cannot migrate into the gel matrix and stuck in the well of the gel. In addition, RP was not observed when CApt1 was omitted (lane 1 and lane 3). This result demonstrated that the aptamer-incorporated circular template was still able to initiate the RCA reaction. The assembled CApt1-GO hybrids were then examined for the RCA reaction in the presence of PDGF, CApt1 or both. There was no RCA reaction when only CApt1 (lane 6) or PDGF (lane 7) was provided. However, RP was observed upon the addition of both CApt1-GO and PDGF (lane 8). All together, these results were in accordance with the proposed mechanism: (1) the strong interaction between DNA and GO prevents the release of the aptamer into the supernatant, thus making it unavailable for the RCA reaction in solution; (2) the presence of cognate target is capable of desorbing the aptamer from the GO surface. We also employed agarose gel electrophoresis to directly monitor RP from the RCA reactions with varying levels of PDGF (Fig. S2). RP can be observed in the presence of PDGF as low as 50 pM.


image file: c9ay01060b-f3.tif
Fig. 3 Analysis of RCA products (RP) by agarose gel electrophoresis. Each reaction was performed for 30 min at 30 °C in 20 μL of 1× RCA reaction buffer containing the indicated components of GO-adsorbed CApt1 (200 nM), TP1 (1 μM), and PDGF (10 nM).

We then investigated the feasibility of performing the RCA reaction on paper. A mixture of PDGF-desorbed CApt1 were incubated with the printed pullulan-encapsulated RCA components (dNTPs and phi29 DNA polymerase) on paper. [α-32P]dGTP was added during the RCA reaction to allow for the radioactive assay. As shown in Fig. 4, in the absence of GO, RP can be observed on paper both with and without PDGF, suggesting that the RCA reaction was effectively carried out on the bioactive paper. In the presence of GO, the RCA reaction was only initiated with the addition of PDGF.


image file: c9ay01060b-f4.tif
Fig. 4 Analysis of RP on a paper array using a radioactivity assay. Each reaction was performed for 30 min at room temperature in 20 μL of solution containing the indicated components of CApt1 and PDGF (10 nM) on TP1-modified paper wells.

It is well-known that a special DNAzyme adopting a G-quadruplex structure can mimic the catalytic function of peroxidase in the presence of hemin.45–48 Thus it can be used for designing various colorimetric or chemiluminescent assays.46 We encoded the antisense sequence of the PW17 DNAzyme into the circular aptamer CApt2.49,50 A mixture of PDGF-desorbed CApt2 were incubated with pullulan-encapsulated RCA components (hemin, dNTPs and phi29 DNA polymerase) on bioactive paper for 30 min at room temperature. After the addition of substrate (containing TMB and H2O2), a strong blue color was observed on the paper. In contrast, no obvious color was generated without the presence of PDGF and CApt2 (Fig. 5a).


image file: c9ay01060b-f5.tif
Fig. 5 (a) Analysis of RP on a paper array using a DNAzyme-based colorimetric assay. Each reaction was performed for 30 min at room temperature in 20 μL of solution containing the indicated components of CApt2 and PDGF (10 nM) on TP2-attached paper. (b) Dose–response curves for PDGF detection with the bioactive paper arrays.

To examine the detection sensitivity of the proposed protocol, we carried out paper-based RCA for the detection of PDGF in 10-fold dilutions (from 0.001 nM to 10 nM). Fig. 5b shows the images of colorimetric responses of the bioactive paper as a function of PDGF concentration. The response curve was obtained by measuring the color intensity using ImageJ for each circular area in the paper well array. It was observed that the intensity gradually increased with increasing PDGF concentrations. A detection limit of 100 pM was obtained on the basis of a signal-to-noise ratio of 3, which was comparable to previously reported electrochemical, colorimetric and fluorescent sensing systems using the linear PDFG aptamer.51–54 It was demonstrated that protein–aptamer interactions can inhibit the DNA polymerase from reading through the aptameric domain.42 If the released PDGF-CApt1 complex was first heated at 90 °C for 5 min before the following paper-based RCA, the system can achieve a detection limit of 10 pM, represents a 10-fold improvement in sensitivity (Fig. S3). We also challenged the proposed assay by analyzing PDGF in a matrix of human serum (Fig. S4). Different PDGF concentrations were spiked in 10% human serum samples and then detected on paper. The calculated recovery values ranged from 92% to 127%, suggesting that our assay could potentially be used for protein detection in biological media.

Conclusions

This work realizes the combination of microscale paper-based analytical devices and rolling circle amplification for colorimetric detection of protein targets. The system uniquely exploits a nanomaterial (graphene oxide), a biorecognition molecule (circular aptamer) and an isothermal amplification method (RCA). Functionalization of graphene nanosensors with circular aptamers enables efficient and specific recognition of proteins. RCA is then performed on paper for amplifying each recognition event into repeating sequence units that can be easily detected by the naked eye. We also made an observation that the circular aptamers exhibited high stability against cellular nucleases in comparison with traditional linear aptamers. Considering the high selectivity, sensitivity and versatility, we envision that the proposed paper-based sensing platform will find useful applications, especially in POC diagnostics.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Funding for this work was provided by the National Natural Science Foundation of China (NSFC; Grant No. 21777013), the Recruitment Program of Global Young Experts (Thousand Talents Program), the Fundamental Research Funds for the Central Universities (DUT17RC(3)040). We thank Dr Yingfu Li and Dr John D. Brennan for assistance with discussing the experimental results.

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Footnote

Electronic supplementary information (ESI) available: Oligonucleotide sequences, dPAGE analysis of digested aptamers in cell lysates, analysis of RCA products by agarose gel electrophoresis, detection of PDGF in human serum samples. See DOI: 10.1039/c9ay01060b

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