Label-free ultra-sensitive colorimetric detection of hepatitis E virus based on oxidase-like activity of MnO2 nanosheets

Characterizations of MnO2 nanosheets

MnO2 nanosheets were synthesized through a facile one step thermal decomposition route. As displayed in Fig. 1, the transmission electron microscopy images of MnO2 nanosheets exhibited a typical two-dimensional layer structure and displayed multiple folds and wrinkled like sheet structure. The high surface-to-volume ratios of ultra-thin sheets could provide large specific surface area and allowing easy contact between reactant molecules and the active sites of nanosheets, thus providing enhanced catalytic activities as well as unique optical properties. High-resolution TEM images (Fig. 1C), shows that obtained nanosheets are free of any particle on surface. The TEM of MnO2 nanosheets at 1 µm scale showing overall view is demonstrated in ESI (Fig. S1) and the size distribution at 200 nm scale in ESI (Fig. S2). From the HRTEM image, the d-spacing was analyzed and found to be around ~ 0.38 nm, which corresponds to the (002) plane displayed in Fig. 1D.

Fig. 1figure 1

TEM image of the MnO2 nanosheets at different scales (A) 1 μm, (B) 200 nm, (C) 100 nm. (D) shows the d-spacing from the HRTEM image of MnO2 nanosheets

The energy-dispersive X-ray (EDS) spectra obtained using TEM microscope shows that Mn and O elements are present in MnO2 nanosheets. The atomic percentage of Mn and O was approximately 25.40% and 74.60%, respectively, as shown in ESI, Fig. S3 and Fig. S4. This confirms successful formation of MnO2 nanosheets.

The crystal structures and phase information of MnO2 nanosheets are acquired from powder X-ray diffraction (PXRD) pattern in Fig. 2A. The diffraction peaks are considerably broadened due to noncrystalline nature of as-synthesized materials. The diffraction pattern observed at 2Ɵ of 11.8°, 24.9°, 36.4°, 41.4°, and 66.6° are corresponding to (211), (301), (600), and (002) crystal planes, respectively, which corresponds to tetragonal MnO2 (JCPDS 44–0141). The minor variations in the distances between nanosheets in various restacked 2D structures can be used to explain the small variations in the (002) diffraction peak angles. By measuring the surface area by N2 adsorption–desorption analysis using a BET analyzer, the results of the surface characteristics studies are shown in Fig. 2B, C, and D. The curves shows the type IV isotherm profiles [31]. Such profiles reveal two types of characteristics of the surface: (1) mesoporous structure and (2) unrestricted multilayer adsorption of the materials. It was found that the surface area of the material was 12.939 m2 g−1. But it was interesting to know that the pore volume was more prominent with an average pore diameter of 19.895 nm. Such a large pore diameter is more suitable for catalytic processes, while the surface area of MnO2 with capture probe and target probe were 19.71 m2 g−1 and 14.76 m2 g−1, respectively, which also confirms the adsorption and desorption of capture and target probes.

Fig. 2figure 2

A X-ray diffraction patterns of the synthesized MnO2 nanosheets. B Nitrogen adsorption and desorption isotherms of the MnO2 nanosheets, C MnO2 with capture probe, and D MnO2 with capture and target probes

To further understand the morphology and adsorption of HEV-DNA on the surface of MnO2 nanosheets, the atomic force microscopy (AFM) was used. From the AFM micrographs (Fig. 3AC), the MnO2 produced from thermal decomposition method shows the root mean square (RMS) surface roughness around 0.0043 µm (Fig. 3A), whereas ssDNA modified MnO2 nanosheets attained a surface roughness 0.1916 µm (Fig. 3B). The change in morphology and surface roughness of MnO2 nanosheets and ssDNA modified MnO2 nanosheets clearly indicates that ssDNA adsorbed on the surface of MnO2 nanosheets. The wavey kind structure was produced on the surface of MnO2 representing the adherence of ssDNA and almost covered the area of the scan (Fig. 3B). Upon the addition of target DNA, it’s interacted with ssDNA on the surface of MnO2 nanosheets. The resulting hybridized DNA released from the surface of MnO2 nanosheets. However, some unhybridized DNA is still adhered on the surface of MnO2 nanosheets which can be seen as wavy kind of appearance on surface of the MnO2 nanosheet as shown in Fig. 3C. The RMS roughness of HEV CP-modified MnO2 + HEV TP is 0.0155 µm which confirm the adsorption and desorption of DNA on MnO2 nanosheets.

Fig. 3figure 3

A AFM images of the MnO2 nanosheets, B MnO2 + HEV CP, and C HEV CP-modified MnO2 + HEV TP

This was further confirmed by zeta potential analysis of all the three materials along with the ssDNA. Unmodified MnO2 nanosheets shows a zeta potential of − 39.6 mV, and ssDNA was − 17.4 mV. After modification with ssDNA, it was found that the zeta potential was increased to − 55.3 mV. Upon adding the target DNA to the system, the hybridized DNA was released from the surface, and the zeta potential was decreased again to − 44.8 mV which is almost closer to the value of unmodified MnO2 zeta potential. This demonstrates that the majority of the ssDNA has been released while a small portion of unhybridized DNA is still remaining on the surface of MnO2 nanosheets Fig. 4A. Furthermore, the HEV capture probe was labeled with Cy3 dye. A 2.5 μM of Cy3-labeled HEV CP was mixed with 10 μg/mL MnO2 catalyst in Na-Ac buffer (pH 3.5, 0.1 M). The fluorescence was recorded in the absence and presence of HEV target probe as shown in Fig. 4B. The bare MnO2 did not show fluorescence, while a high fluorescence was observed for MnO2-CP demonstrating that MnO2 is successfully modified with HEV DNA. But when HEV target was added into the system, the target probe hybridized with capture probe and released from the surface; hence, a decrease in the fluorescence was observed.

Fig. 4figure 4

Zeta-potential analysis of A MnO2 nanosheets, ssDNA, MnO2-CP and in the presence (MnO2-CP-TP) of the HEV target probe. B Fluorescence spectra of MnO2 nanosheets, MnO2-CP and presence of the HEV target probe (MnO2-CP-TP)

Proof of concept and verifications

In the presence of DNA, the surface of MnO2 nanosheets can be masked that inhibits the biomimetic oxidase-like activity of MnO2 nanosheets. DNA affects the catalytic activity of the MnO2 nanosheets; it adsorbs on the surface of MnO2 nanosheets via the negatively charged phosphate backbone [32] and by van der Waals force of interaction between the surface of MnO2 material and nucleobases of DNA [33] and, thus, reasonably inhibits the oxidase-like activity of MnO2 [34]. Encountering the HEV target DNA, the complementary target ssDNA is hybridized with the capture ssDNA probe, between the densely negatively charged phosphate backbones, the nucleobases are buried, and double-strand DNA (dsDNA) conformation is produced. By employing conserved sequences, no cross-reactivity existed because of specific complementarity. The dsDNA conformation can reduce the intensity of the Van der Waals interaction between dsDNA and the MnO2 surface and inhibit interactions between nucleobases and material surfaces [35]. As a result of the electrostatic repulsion between dsDNA and MnO2, the system’s oxidase activity will recover as dsDNA is released from the surface of MnO2 (Scheme 1). The basic principle behind the colorimetric sensing of HEV is based on the biomimetic oxidase-like activity of MnO2 nanosheets. As shown in Fig. 5A, the TMB was colorless with no characteristic absorbance from 350 to 800 nm. After adding the as-prepared MnO2 nanosheets, the oxidation of the colorless TMB produced a blue-colored product (oxTMB) as can be seen in the inset of Fig. 5A and giving a high absorbance peak. However, after the addition of the HEV capture probe into the MnO2-TMB system, the absorbance at 652 nm was decreased by hindering the oxidation of TMB to oxTMB shown in Fig. 5B. This can be attributed to the adsorption of the HEV capture probe on the basal plane of MnO2 through the negatively charged phosphate backbone [34]. The oxidation of TMB to oxTMB would be reduced as a result of covering the surface area of MnO2 nanosheets, leading to the inhibition of biomimetic activity. Consequently, the intensity of the 652 nm absorbance reduced. However, there is some incomplete surface coverage of captured ssDNA on MnO2 nanosheets for catalytic reaction which gives absorbance peak at 652 nm. But, upon the addition of target DNA to the MnO2-TMB-HEV-CP system, the target probe binds to the capture probe on the surface of MnO2 because of DNA hybridization. Hence, the hybridized DNA from the surface of MnO2 is released, and the oxidase-like activity of the MnO2 is recovered. Confirmation of the feasibility of the target sensing approach through visual observations can be seen in ESI, Fig. S5. Without the use of H2O2, this efficient reaction between the TMB substrate and MnO2 nanosheets offers tremendous stability and reproducibility. These results established the practicality of this colorimetric sensing mechanism and applied it to the detection of HEV target probe.

Scheme 1scheme 1

Schematic diagram of MnO2-TMB system for the colorimetric sensing of HEV

Fig. 5figure 5

UV–visible absorption spectra of (A) only TMB, only MnO2 and TMB + MnO2. Inset of A is the visual observations. (B) The mixtures (TMB, MnO2, TMB + MnO2, TMB + MnO2 + HEV CP and TMB + HEV CP-modified MnO2 + HEV TP) in the presence of TMB (1 mM) and MnO2 (20 μg/mL)

Optimization of assay conditions

For the highly throughput results of the HEV detection sensor, the critical parameters such as pH, MnO2, and HEV capture probe were studied systematically to establish the optimal conditions. The concentration of MnO2 was investigated to determine the optimum concentration for high oxidase-like activity. Different MnO2 concentrations (0, 10, 20, 40, and 60 μg/mL) were added to MnO2-TMB reaction system. In ESI, Fig. S6A depicts that the UV–visible absorbance peak intensity centered at 652 nm increases with increasing concentrations of MnO2. There was a linear relationship between MnO2 concentration and absorption spectra at 652 nm with the regression coefficient (R2 = 0.999) as shown in ESI, Fig. S6B. For high accuracy, we employed 10 μg of MnO2 for further experiments of HEV detection.

As the pH value is critical for the high oxidase-like activity of MnO2, Na-Ac buffer of pH range from 3 to 6 was studied. As presented in ESI, Fig. S7, the absorbance spectra of the reaction system decreased steadily with increasing pH value. MnO2 exhibits high enzymatic activity in mildly acidic conditions, and MnO2 nanosheets have a high oxidation capability [36]. So, Na-Ac buffer of pH 3.5 was selected for further experiments.

Adsorption of HEV-DNA on MnO2 nanosheets

To investigate the application of MnO2-TMB sensing platform for the detection of HEV, the system was first applied to investigate the adsorption of HEV capture probe on MnO2 surface. Under the optimum experimental conditions, different concentrations of HEV capture probe in the range of 0–2.5 μM were applied to adsorb on the surface of MnO2. The concentrations of HEV capture probe have a huge effect on the catalytic activity of MnO2. As the concentration of HEV capture probe increased from 0 to 2.5 μM, the absorbance spectra at 652 nm decreased as illustrated in Fig. 6A. The color of the reaction was considerably changed from dark blue to light blue which was also confirmed by visual observations shown in Fig. 6B. The HEV capture probe was physically adsorbed successfully by MnO2 via phosphate backbone of DNA which is often bound by some nanoparticles [37]. In literature, it is reported that due to large number of terminal phosphate groups, its binding affinity to nanoparticles is higher [38]. DNA affects the catalytic activity of the nanomaterial; it adsorbs on the surface of the nanomaterial via the negatively charged phosphate backbone [32]. This indicates that the adsorption of HEV capture probe masked the surface of MnO2, so less MnO2 surface is available for the catalytic oxidation of TMB and hence decreases the absorbance spectra at 652 nm. When the concentration of HEV capture probe increased, the absorbance spectra at 652 nm decreased. Figure 6C shows that there is an indirect relationship between the change in absorbance and the HEV capture probe concentration from 0 to 2.5 μM with a correlation coefficient of 0.984. Figure 6D shows a clear difference in the absorbance spectra of TMB + MnO2 and TMB + MnO2 + HEV CP.

Fig. 6figure 6

UV–visible absorption spectra of A varied concentrations (0, 0.5, 1.5, and 2.5 μM) of DNA adsorption on MnO2 in presence of TMB (1 mM) and MnO2 (20 μg/mL). B shows the visual representations of adsorption of different concentrations of DNA on MnO2 resulting in decrease oxidation of TMB. C The liner calibration between the different concentrations of DNA adsorption on MnO2 and the decrease in TMB oxidation. D UV–visible absorption spectra of TMB + MnO2 sensing system in the absence (black) and presence (red) of adsorbed DNA on MnO2

Analytical performance of the sensor for HEV detection

The proposed colorimetric sensor was also employed to detect HEV under the ideal experimental conditions. With increasing the concentration of HEV target probe ranging from 1 fM to 100 nM, the absorbance spectra at 652 nm of the sensing platform was gradually increased as displayed in Fig. 7A. As the concentration of HEV target probe increases, more number of target probes bind with capture probe, and the surface of MnO2 is unmasked. Hence, the oxidase-like activity of the MnO2 is recovered by the oxidation of TMB to oxTMB. The absorbance spectra at 652 nm are intensified. Figure 7B shows the visual confirmation of the increased oxidation of TMB by MnO2. With the increasing concentration of target probes in the sensing system, more capture probes are released from the surface of MnO2. This confirms more availability of MnO2 for the oxidation of TMB and the color of the sensing system changes from light blue to dark blue. Figure 7C illustrates a linear relation of change in absorbance spectra and the target probe concentration from 1 fM to 100 nM with the regression coefficient of 0.998. The absorbance spectra at 652 nm were directly related to the sensing of HEV; this correlates linearly with the concentration of HEV target probe. The LOD was calculated to be 3.26 fM based on the standard deviation of the response (Sy) of the curve and the slope of the calibration curve (S) at levels approximating the LOD according to the formula: LOD = 3.3(Sy/S). The LOQ was calculated to be 36.08 fM on the basis of standard deviation of the response (SD) and the slope of the calibration curve (S) according to the formula: LOQ = 10(Sy/S). The standard deviation of the response was determined based on the standard deviation of y-intercepts of the regression lines. The results are comparatively better than those of earlier reports tabulated in Table S1 in ESI. There is an obvious change in the absorbance spectra of TMB + MnO2 + HEV CP and TMB + HEV CP-modified MnO2 + HEV TP. In TMB + MnO2 + HEV CP sensing system, most of the MnO2 surface is hindered by the adsorbed capture probe, so fewer MnO2 active sites are available to catalyze the TMB present in the reaction system. This can also be attributed to the presence of bigger pore size and volume. These can also provide necessitated active sites possible to even load the ssDNA to MnO2, and this is evident from BET analysis. As the target probe is added into the system, the capture probe binds with the target probe because of their complementarity with each other. Hence, the oxidase activity of MnO2 is recovered for the oxidation of TMB as shown in Fig. 7D.

Fig. 7figure 7

A UV–visible absorption spectra for detection of different concentrations (0, 1 fM, 100 fM, 1 pM, 100 pM 1 nM, and 100 nM) of HEV target in presence of TMB (1 mM) and MnO2 (10 μg/mL). B Visual illustrations of detection of different concentrations of HEV target probes leading to recovery of oxidase-like catalytic activity of MnO2. C Displays the linear calibration of detection of HEV target probes and recovery of oxidase-like activity of MnO2. D UV–visible absorption spectra of TMB-MnO2 sensing system in the absence (red) and presence (black) of HEV target probe and 20 μg/mL of MnO2 catalyst

Stability and selectivity of the biosensor

In order to assess the reliability of the developed sensor, the cyclic stability tests of MnO2 and MnO2 + HEV-CP were performed at a 1-week interval for a period of 1 month as displayed in Fig. 8A. It indicates that our prepared material exhibits good catalytic activity and stability over a month without affecting its primary performance. The selectivity of this sensing system was subsequently studied by monitoring the change in absorbance activity with other interfering molecules. To assess the specificity of the suggested sensor, we selected other mismatched DNA sequences such as IBV, HBV, HCV, IAV, HAV, HEV-3, and HEV-4 as shown in Table S2. The concentrations of these interferents were set as 1 pM, while 100 fM concentration was set for HEV target (equivalent to 0.1 pM). As shown in Fig. 8B, due of the non-specific interaction with the HEV capture probe, the biosensor responses to the other virus sequences are much lower, even though they were in higher concentration than HEV. This makes our developed sensor unique to the target analyte. This confirms that our developed colorimetric sensor possesses excellent specificity and selectivity for HEV detection.

Fig. 8figure 8

Stability and selectivity of the developed sensor. A Stability of oxidase activity of MnO2 nanosheets and hindering oxidase activity of MnO2 nanosheets by HEV-CP in the presence of TMB (1 mM), MnO2 (20 μg/mL), and HEV-CP (2.5 μM). B Selectivity of HEV target (100 fM) over potential interferences. IBV, HBV, HCV, IAV, HAV, HEV-3, HEV-4 (1 pM). The incubation time selected was 5 min. Error bar represents the standard deviation for three determinations

Analytical performance in spiked serum samples

To further explore the analytical performance of our colorimetric sensor in complex biological samples, the human serum obtained from Sigma-Aldrich was diluted tenfold after centrifugation. Then, the diluted serum sample was combined with 10 μL of various HEV DNA concentrations (100 fM, 1 pM, 100 pM, 1 nM, and 100 nM) and incubated for 30 min at 37 °C. To examine whether the developed sensor could be applied for the sensing of HEV in a real sample, the absorbance responses are tested in spiked serum samples. As depicted in Fig. 9A, the biosensor responses display a similar tendency of increasing absorbance with increasing concentrations (100 fM–100 nM) of HEV targets as in the buffer. The LOD and LOQ were calculated to be 9.32 fM and 28.24 fM, respectively. Figure 9B shows a linear relation of change in absorbance spectra vs different concentrations of target HEV (100 fM, 1 pM, 100 pM, 1 nM, and 100 nM) with the regression coefficient value of 0.99 (n = 3). The absorbance spectra of target detection in Na-Ac buffer and human serum are shown in ESI, Fig. S8. The recoveries of HEV target DNA ranged from 97.5 to 101.5% tabulated in Table 1, proving that the suggested method may be used to diagnose HEV in patients without the need for complex pretreatments.

Fig. 9figure 9

Sensor performances for different concentrations of HEV analytes in serum. A UV–visible absorption spectra of detection of different concentrations (100 fM, 1 pM, 100 pM, 1 nM, and 100 nM) of HEV target in serum in presence of TMB (1 mM) and MnO2 (10 μg/mL). B depicts the linear calibration of detection of HEV target in human serum samples

Table 1 Detection of HEV target in serum sample with this sensor

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