An ultrasensitive aptasensor of SARS-CoV-2 N protein based on ion current rectification with nanopipettes

Abstract

Since the outbreak of COVID-19 in the world, it has spread rapidly all over the world. Rapid and effective detection methods have been a focus of research. The SARS-CoV-2 N protein (NP) detection methods currently in use focus on specific recognition of antibodies, but the reagents are expensive and difficult to be produced. Here, aptamer-functionalized nanopipettes utilize the unique ion current rectification (ICR) of nanopipette to achieve rapid and highly sensitive detection of trace NP, and can significantly reduce the cost of NP detection. In the presence of NP, the surface charge at the tip of the nanopipette changes, which affects ion transport and changes the degree of rectification. Quantitative detection of NP is achieved through quantitative analysis. Relying on the high sensitivity of nanopipettes to charge fluctuations, this sensor platform achieves excellent sensing performance. The sensor platform exhibited a dynamic working range from 10²-10⁶ pg/mL with a detection limit of 73.204 pg/mL, which showed great potential as a tool for rapidly detecting SARS-CoV-2. As parallel and serial testing are widely used in the clinic to avoid missed diagnosis or misdiagnosis, we hope this platform can play a role in controlling the spread and prevention of COVID-19.

Graphical Abstract

SARS-CoV-2 N protein cause changes in the degree of ion rectification effect.

1. Introduction

At the end of 2019, COVID-19 spread to the whole world and caused a large number of casualties [1]. SARS-CoV-2, which causes severe acute respiratory syndrome, is the infectious agent responsible for this pandemic [2]. SARS-CoV-2 spreads from person to person through virus-containing droplets expelled when an infected person coughs, sneezes, talks, and breaths [3]. Symptoms include fever, dry cough, headache, and so on [4].

The rapid diagnosis of COVID-19 is vital for halting the virus’s spread, and it plays a key role in resuming flights, promoting the development of tourism, and normalizing economic activities. Nowadays, RT-PCR is primarily used for the clinical detection of SARS-CoV-2, while antibody-targeted ELISA is used for serological detection. Detection of SARS-CoV-2 specific antibodies is an alternative diagnostic technique. After 5–7 days of infection, the IgM antibody was discovered, and then IgG was detected [5]. It has a clear latency when compared to PCR detection and is typically employed as a backup method.

SARS-CoV-2 has a single-stranded RNA genome that is 30,000 nucleotides in length [6] . SARS-CoV-2 encodes four structural proteins in its RNA genome: the spike (S), membrane (M), envelope (E), and nucleocapsid (N) [7]. The majority of currently used PCR-based techniques have a detection limit of 100 copies/mL or less, which corresponds to viral detection 2–3 days before symptoms appear [8]. Although very effective as the “gold standard” for SARS-CoV-2 identification, RT-PCR requires trained personnel, pricey equipment, and time-consuming sample preparation and analysis [9]. At present, some RNA detection methods have also been proposed, including RT-LAMP [10], [11], CRISPR [12], [13], and so on. These methods have their advantages over PCR, but all require sophisticated instruments, expensive reagents, and skilled instrument operators.

Meanwhile, RNA is less stable during transport and storage than protein, improper storage of RNA and improper sample handling is easy to cause false-negative results in final detection [14]. As the most expressed virus-related protein during infection [7], the detection of N protein (NP) is significant for the identification of SARS-CoV-2. Different from RNA, the detection of protein doesn’t involve the amplification process, which avoids false negatives caused by the false replication generated during the nucleic acid amplification process. Since no amplification process is involved, direct detection of proteins poses a huge challenge. Researchers have proposed electrochemical sensors [15], [16], fluorescent sensors [17], and chemiluminescence methods for the detection of NP [18].

Electrochemical detection is frequently used and benefits from quick analysis, low detection limits, and high sensitivity [19]. In 1997, ion current rectification (ICR) was found in nanopipette experiments [20]. The effect of ion current rectification was observed as asymmetric current-voltage (I–V) curves, with the current recorded for one voltage polarity higher than that for the same absolute value of the voltage of opposite polarity. Siwy referred that the ICR has an intimate relation to the charge of the inner wall of a nanopipette [21]. Changes in steric hindrance and charge density on the nanopipettes’ inner surface will have an impact on ion transit, as well as a quick electrochemical reaction. Based on this, the researchers constructed a nanopipette sensor based on ICR. According to the electrochemical characterization changes caused by the change of the surface charge of the nanopipettes before and after the capture probe captures the target are observed, the qualitative and quantitative analysis of the target is realized. This type of sensor has been used to detect nucleic acids [22], [23], [24], proteins [25], [26], [27], enzymatic activities [28], metal ions [29], [30], [31], etc.

The selection of the capture probe is important for the construction of the sensor. Some specific recognition targets based on NP detection use molecular imprinting [32], [33], antigen-antibody reactions [34], [35], [36], [37], [38], and aptamers [15]. Aptamers display more affinity and more binding sites with lower molecular weight and are easy to synthesize/modify, and cost-effective. Benefit from the strong negative charge of the aptamer, the signal is pronounced when the immobilized aptamer captures the target allowing one to identify the captured single molecule [39]. Therefore, aptamers were prefer chosen for modifying nanopipettes. Aptamers have so far been demonstrated to be capable of binding to a variety of targets, such as metal ions [40], [41], proteins [42], [43], [44], and even cells [45]. Researchers have screened aptamer by SELEX technique and constructed aptasensor for the detection of SARS-CoV-2 S1 protein [25].

In this research, we proposed a sensor based on ICR to detect NP. Aptamer-functionalized nanopipette is used for specific capture and detecting NP. The aptamer was introduced to the nanopipette via a covalent modification process. At the pH used for detecting NP, the aptamer was negatively charged while the NP (pI=10.07) was positively charged. The biospecific interaction of aptamers with proteins causes a decrease in charge density and an increase in steric hindrance. Coupled with an electrochemical technique, the protein can be sensitively identified. The sensor shows good resistance against interference thanks to its high affinity, low limit of detection (LOD), and good stability. As expected, the sensor can play a role in assisting other COVID-19 detection methods. ( Fig. 1).

Fig. 1.

Schematic illustration of the aptamer-functionalized nanopipettes for detection of N protein. (A) Schematic diagram of the detection device. (B) N protein was polymerized onto the inner surface of nanopipettes via a specific interaction with aptamers which can neutralize the negative surface charge. (C) Schematic description of the covalent modification procedures of nanopipettes.

2. Experimental section

2.1. Nanopipette fabrication and characterization

Before pulling the nanopipette with capillaries (OD 1.0 mm, ID 0.58 mm), they should be cleaned with piranha solution (98% H₂SO₄/30% H₂O₂, V/V=3:1) in 80 ℃ for 2 h and rinsed by DI water. The capillaries were then dried in a stream of nitrogen. After being dried, capillaries were pulled by P-2000 (Sutter Instruments, Novato, CA) through a program as follows: Heat 325, Filament 1, Velocity 10, Delay 145, and Pull 180. The puller should be preheated for at least 15 min.

After nanopipettes were prepared, they were characterized by a JSM-7800 F scanning electron microscope (JEOL) at 5 kV accelerating voltage (Fig. S1A). The inner diameter was estimated using the I-V curves observed in a solution of 1 M KCl.

2.2. Modification of nanopipette

After treatment with piranha solution, the nanopipettes finished silicon hydroxyl terminal modifications. Firstly, the nanopipettes were filled with 5% (V/V) 3-aminopropyl-triethoxylsilane (APTES) solution in ethanol and the tips of the nanopipettes were immersed in the same solution in the dark for 1 h. Afterward, the nanopipettes were washed with ethanol and baked at 120 ℃ for 1 h subsequently. The nanopipette was then treated with PB (10 mM K₂HPO₄, 10 mM KH₂PO_4, and 1 mM KCl, pH7.4) containing 2.5% (V/V) glutaraldehyde overnight, followed by rinsing with PB thoroughly. The aldehyde-functionalized nanopipettes were connected with 0.6 μM aptamer for 120 min at room temperature. Finally, the nanopipettes were rinsed with PB and ready for use.

Before aptamer-functionalization, the aptamer should be heated at 95 ℃ for 5 min and cooled down to uncoil the DNA.

2.3. Electrochemical measurements

The I-V curves were measured by CHI760E (Shanghai CHI Instrument Co. Ltd., China). Linear sweep voltammetry (LSV) was tested in PB. The scan voltage was adjusted to range from − 1.0 to + 1.0 V with a 0.05 V/s scan rate, and the pulse amplitude was set to 0.001 V. All measurements were performed at room temperature. The bulk solution and the infilled solution were kept the same throughout the experiments. The rectification ratio r (r = log₂ |I^-/I⁺|, I^- is the current in −1 V and I⁺ is the current in +1 V) can illustrate the charge status of the nanopipettes. When r < 0, the inner surface charge of nanopipettes is positive while r > 0 the charge in the inner wall is negative. A normalized current change (△I/Io, △I I-Io, Io and I represent the current before and after the incubation at −1 V) was used to reduce the current variation between different nanopipettes.

Different concentrations of NP are prepared in PB and used for the measurement of the respective I–V curves.

3. Results and discussion

3.1. Nanopipettes characterization

The orifice diameter was approximately 125 nm according to the SEM picture (Fig. S1A), which was similar to the calculated value from the I-V curves measured in 1 M KCl (Fig. S1B) using the equation below [46], [47].

$$\mathrm{R}=\frac{\mathrm{\gamma }\cot \mathrm{\theta }/2}{\mathrm{\pi }\mathrm{a}}$$

where R is the resistance of the nanopipettes, γ is the electrolyte’s resistivity (S/m), $\mathrm{a}$ is the radius of the nanopipettes, and θ is the cone angle (9° in this example).

3.2. Modification of nanopipettes

It is well understood that nanopipettes’ inner surface modification has a significant impact on the mass and charge transport parameters [21]. The surface charge changes can be characterized using the I-V curves ( Fig. 2A) and rectification ratios (Fig. 2B). Compared to the commonly used electrochemical impedance spectroscopy and cyclic voltammetry, LSV can directly obtain the current characterization related to rectification effects by virtue of simpler parameter settings.

Fig. 2.

Nanopipette modification processes. (A) Monitoring the process of chemical alteration on the inner surface of the nanopipettes by measuring the I-V curves in PB. (B) ICR ratio (r-value) of the modification stages (silica hydroxyl groups, amino groups, aldehyde groups, and immobilized aptamer strands (0.6 μM), respectively).

At physiological pH, the dissociation of silanol groups makes silica surfaces acquire a charge in contact with the solution[48].

$$\mathrm{SiOH}\rightleftharpoons {\mathrm{OH}}^{-}+{\mathrm{H}}^{+}$$

Silicate glass surfaces immersed in water are known to have a negative surface charge density in this scenario. According to the current of an undecorated nanopipette, the measured current for negative voltage is higher than that of positive voltage (Fig. 2A) and an r-value of 1.215 ± 0.185 (Fig. 2B) which suggested the nanopipettes’ inner surface was negatively charged. After APTES modification, the r-value was converted to − 3.043 ± 0.190 (Fig. 2B) and the nanopipettes’ inner surface was positively changed which can be characterized by I-V curves (Fig. 2A). On one end of APTES are three ethoxy silanes that can covalently bond to silica surfaces after undergoing a condensation reaction. A primary amine group on the other end is protonated in aqueous solutions (pI=9.6). Therefore, an APTES-covered surface will be positively charged at pH7.4 [49].

Aldehyde groups were introduced to the nanopipettes by crosslinking glutaraldehyde with the fixed amino groups. Because the aldehyde group is neutral, the current of positive voltage is close to the current of negative voltage (Fig. 2A) and the changed nanopipette had an r-value of − 0.188 ± 0.047 (Fig. 2B), indicating a significantly lower positive charge than the APTES modified nanopipettes.

Upon immobilizing aptamer, the nanopipettes exhibited an r-value of 3.039 ± 0.201 (Fig. 2B), which showed that the inner wall of the nanopipettes had accumulated more negative charges and resulted in a higher current at a negative voltage (Fig. 2A) that the phosphate backbones of DNA were deprotonated in a neutral state [26].

All of the I-V curves (Fig. 2A) and r-values (Fig. 2B) can demonstrate the success of nanopipette modification.

3.3. The influence of experimentation conditions on sensing performance

The biosensor’s detection performance is significantly influenced by the experimental settings. As the probe is used to capture the targets, the concentration of the aptamer could have a substantial impact on how well the detection works. Different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, and 1 μM) were chosen to explore the optimal concentration and the incubation time was 120 min ( Fig. 3A, B). When the concentration of aptamer was higher than 0.6 μM, the I-V curves changed slightly, indicating that the binding of aptamer onto the nanopipettes had reached saturation, so we concluded that 0.6 μM aptamer was optimal for modification nanopipettes. At too high concentrations, the aptamer might cause steric hindrance. After that, the incubation time (30, 60, 90, and 120 min) of probes and NP (100 ng/mL) was studied to shorten the detection time and increase the accuracy of the results. It was observed that the I-V curves were stable after 60 min of incubation and the current change was enough to be found, so 60 min was determined as the incubation time (Fig. 3C, D).

Fig. 3.

Optimization of experiment parameters. (A) I-V curves of the nanopipettes for optimizing the aptamer concentration (0.1, 0.2, 0.4, 0.6, 0.8, and 1 μM). (B) Normalized ionic current changes of different concentrations of aptamers at the bias voltage of − 1 V. (C) I-V curves of the nanopipettes for optimizing the N protein capture time (30, 60, 90, and 120 min). (D) Normalized ionic current changes of different N protein capture time at the bias voltage of − 1 V.

3.4. Detection of the NP

The working principle of the sensor platform can be explained by the accumulation and dissipation of electric charge. Under neutral conditions, the surface of aptamer-functionalized nanopipettes is negatively charged, with cation selectivity, and it generates an electric current driven by an electric field. When the NP (pI=10.07) is captured by the aptamer, the positively charged NP will neutralize the negative charges, which will reduce the density of the surrounding cations and the current generated. We use standardized current change ( Fig. 4B) to judge the relationship between NP concentration and current response.

Fig. 4.

Quantitation of the N protein using the aptamer-functionalized nanopipettes. (A) I-V curves of the nanopipettes for detecting different concentrations of the N protein. (B) Normalized ionic current changes of different concentrations of the N protein at the bias voltage of −1 V. (C) Normalized ionic current change vs different N protein concentrations. (D) Normalized ionic current changes vs the logarithm of the N protein concentration ranging from 100 pg/mL to 1 μg/mL.

To determine the analytical range, different concentrations (10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ pg/mL) of NP were incubated with the aptamer-functionalized nanopipettes at room temperature for 60 min, followed by the I-V measurement (Fig. 4A). As depicted in Fig. 4B, the normalized current change enhanced with the increase of NP concentration and showed a good linear relationship range from 10² to 10⁶ pg/mL (Fig. 4D). The linear relationship for NP is △I/Io= 0.255–0.187lgC with R² = 0.992 and the LOD was down to 73.204 pg/mL, which exhibits better sensing performance than other reported NP detection methods as shown in Table S1.

3.5. Specificity of the nanopipette sensor

Target-free sensors using aptamer capture analysis are susceptible to electrostatic and other non-specific bindings. The aptamer-functionalized nanopipettes were used to detect IgM (1 μg/mL), BSA (1 μg/mL), Thrombin (1 μg/mL), and a mixture of the same concentration at room temperature for 1 h to evaluate the specificity. As shown in Fig. 5, compared with 1 ng/mL of NP, the effect of a high concentration of protein on rectifying degree is almost negligible, which is due to the high specificity of aptamer and NP recognition. The experimental results show that the nanopipette sensor platform has good specificity, and the nonspecific interference can be ignored.

Fig. 5.

Specificity and anti-interference ability test of aptamer-functionalized nanopipettes. (A) I-V curves for detection of BSA (1 μg/mL), IgM (1 μg/mL), Thrombin (1 μg/mL), the mixture of them, and NP (1 ng/mL). (B) Corresponding normalized ionic current changes.

3.6. Stability of the nanopipette sensor

To further explore the possibility of developing and manufacturing sensors in clinical diagnosis and biological detection, it is of great significance to study the stability of sensors. The nanopipette sensor based on aptamer functionalization was stored at 4 ℃ for 7 days, and the rectification performance of the nanopipette was tested by LSV, in which the concentration of NP was 1 μg/mL. As shown in Fig. 6, the response of the nanopipettes to the rectification change of 1 μg/mL NP did not change obviously after 7 days.

Fig. 6.

Stability of the nanopipette aptasensor for detecting N protein within 7 days. The stability of the aptamer-functionalized nanopipettes was characterized by the normalized ionic current changes under the bias voltage of − 1 V within 0.6 μM aptamer functionalized and 1 μg/mL N protein reacted surface in 7 days.

3.7. Real sample analysis

To verify the applicability of the sensor platform, the potential of the developed aptamer functionalized nanopipettes in actual samples was studied by detecting the NP added in human serum with different concentrations. Different concentrations of NP (0.01 μg/mL, 0.1 μg/mL) were added to the serum diluted by PB for 10 times, and the presence of NP was detected by the nanopipette sensor. According to the results obtained in Table S2, the sensor platform obtained recovery rates of 104% and 106% in two NP samples (0.01 μg/mL, 0.1 μg/mL) which indicated that the constructed nanopipette sensor showed high reproducibility, accuracy, and feasibility in detecting NP in human serum.

4. Conclusions

In this research, we have developed the NP sensor platform for the rapid and sensitive detection of NP in serum based on the unique ion rectification effect of nanopipettes. By functionalizing the inner wall of the nanopipette with aptamers, the platform takes full advantage of the high sensitivity of the nanopipette to surface charge fluctuations and the high specificity of aptamers for target recognition which can achieve highly specific capture of NP, exhibiting an extremely low detection limit of 73.204 pg/mL within a broad linear NP concentration range from 100 pg/mL to 1 μg/mL and strong specificity for NP in complex samples with good stability. Moreover, the method is short in time, low in cost and simple in operation. The developed sensor is based on a completely different approach to NP detection compared to the currently constructed approach to NP detection which is expected to play a role in COVID-19 detection. By altering the modified capture probe we anticipate that the sensitive detection capability, stability, simplicity and inexpensive fabrication will make nanopipettes a universally useful platform for epidemic prevention and control.

CRediT authorship contribution statement

Conceptualization: Wenhao Ma, Wanyi Xie, Changjun Hou, Danqun Huo, Deqiang Wang, Methodology: Wenhao Ma, Wanyi Xie, Rong Tian, Xiaoqing Zeng, Liyuan Liang, Writing – original draft: Wenhao Ma, Wanyi Xie, Funding acquisition: Changjun Hou, Danqun Huo, Deqiang Wang, Supervision: Changjun Hou, Danqun Huo, Deqiang Wang.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Wenhao Ma is presently a Ph.D. student in College of Bioengineering at Chongqing University. His research interests are nanopore sensing.

Wanyi Xie is presently a Ph.D. student in Chinese Academy of Sciences. Her research interests involve optoelectronic sensing of nanopores.

Rong Tian received her Ph.D. from Nanjing University of Technology in 2015. In 2016, she was engaged in postdoctoral research at Suzhou Institute of Nanotechnology, Chinese Academy of Sciences and Tsinghua University, respectively. She works on single-molecule biosensors.

Xiaoqing Zeng is presently a Ph.D. student in Chinese Academy of Sciences. She works on nanopores to detect HIV markers.

Liyuan Liang received her Doctor of Science degree from the University of Bordeaux, France in 2011 and joined the Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences in 2014. She studies the molecular dynamics of molecular perforation in solid-state nanopores

Changjun Hou completed his Ph.D. in biomedical engineering in 2004 at Chongqing University. After a period of research at the University of Illinois at Urbana Champaign as a visiting scholar, he returned to China and got tenure at the college of bioengineering at Chongqing University. His scientific interests rest on the design and construction of biochemical sensing systems based on porphyrins and other sensitive materials.

Danqun Huo received her Ph.D. in biomedical engineering in 2004 at Chongqing University and has been a professor in biomedical engineering. Her research interests include the development and utilization of biological resources, research on new technologies for biological detection, nanomaterials, and biochips.

Deqiang Wang received his Ph.D. from the Institute of Microelectronics, Chinese Academy of Sciences in 2006. From 2007 to 2010, he was engaged in postdoctoral research on natural nanopores and solid nanopores at the University of Texas at Arlington (UTA) and the University of Illinois at Urbana-Champaign (UIUC). His research interests are nanopore single-molecule sequencing and sensing technology, and novel micro- and nano-fabrication technologies.

Appendix A. Supplementary material

Supplementary material

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Data Availability

Data will be made available on request.