key: cord-0933579-c85w20sg
authors: Nasrin, Fahmida; Chowdhury, Ankan Dutta; Ganganboina, Akhilesh Babu; Achadu, Ojodomo J.; Hossain, Farzana; Yamazaki, Masahito; Park, Enoch Y.
title: Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes
date: 2020-11-25
journal: Mikrochim Acta
DOI: 10.1007/s00604-020-04656-2
sha: 1403c5db3adc5c56ab03de6740b0298f4b36ea28
doc_id: 933579
cord_uid: c85w20sg

The critical goal of sensitive virus detection should apply in the early stage of infection, which may increase the probable survival rate. To achieve the low detection limit for the early stage where a small number of viruses are present in the sample, proper amplified signals from a sensor can make readable and reliable detection. In this work, a new model of fluorescent and electrochemical dual-mode detection system has been developed to detect virus, taking recombinant Chikungunya virus E1 protein (CHIK-VP) as an example. The hydrophobic quantum dots (QDs) embedded in the lipid bilayer of liposome and methylene blue (MB) encapsulated in the inner core of liposomes played a role of dual-signaling modulator. After CHIK-VP addition, the nanocomposites and APTES-coated Fe(3)O(4) nanoparticles (Fe(3)O(4) NPs) were conjugated with antibodies to form a sandwich structure and separated from the medium magnetically. The nanoconjugates have been burst out by chloroform as surfactant, and both the QDs and MB are released from the liposome and were then monitored through changes in the fluorescence and electrochemical signals, respectively. These two fluorometric and electrochemical signals alteration quantified the CHIK-VP in the range of femtogram to nanogram per milliliter level with a LOD of 32 fg mL(−1), making this liposomal system a potential matrix in a virus detection platform. [Figure: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00604-020-04656-2.

Virus detection with higher sensitivity and selectivity, along with the presence of other interferences, is of great importance to control the annual epidemic [1] [2] [3] [4] [5] . In most viral diseases, the progress of vaccination is a time-consuming process that encourages the necessity for the development of rapid sensing technologies to prevent the viral outbreak. The rapid Fahmida Nasrin and Ankan Dutta Chowdhury contributed equally to this work.

identification and quantification of viruses in all possible samples are of great significance for prompt treatment and effective management of illness [6] [7] [8] . Conventional virus detection methods like polymerase chain reaction (PCR) and other branched-chain DNA-based methodologies are not well suited for point-of-care diagnosis [9, 10] . These are time-consuming and expensive and require intensive sample preparation with high skilled personnel [11] [12] [13] . Therefore, the development of an alternative method of virus detection is in high demand, detecting the viruses in its early stage of infection with high sensitivity and performing real-time monitoring purposes.

Target amplification is employed using the liposomal matrix to attain high sensitivity, which has emerged in the last decade as an attractive approach where a target virus bound to a single liposome can generate amplified signals by releasing the encapsulated signal probes [14] [15] [16] [17] . Therefore, a small number of viruses can generate an intense signal from the liposome's encapsulated probes [18] [19] [20] [21] . Moreover, the encapsulated signal probes can be protected inside the liposome until the external triggers appear and significantly reduce the background noise, which is the great advantage of using liposome-based systems. However, in a real-time application, to attain the detectability of the virus at a very low concentration in the sample medium where a lot of impurities can interfere with the sensing, it is essential to purify the target virus from its medium. To achieve this, the well-established Fe 3 O 4 magnetic nanoparticles can be extremely useful for removing the interferences from the virus-loaded liposomal platform after proper modification [22] [23] [24] [25] [26] [27] [28] [29] . The magnetic nanoparticles' application is quite common in recent literature due to its facile synthesis, APTES modification, and the high magnetic moment [26, 30, 31] . Though there are some advantages of encapsulated probes in the liposomal matrix for amplification of signals, from previous reports, it can be noted that the reliability of the detection remains questionable, especially in low concentration range [26, 31] . Therefore, a successful combination of the liposomal matrix with magnetic nanoparticles can be applied to construct a new class of the biosensor where multiple detections can strengthen the results.

Energized by few recent reports on liposomal amplification and magnetic separation, in this study, a dual-functional signal amplification system containing fluorescent quantum dots (QDs) embedded and methylene blue-encapsulated liposomes (QDs-liposome@MB) has been synthesized for the detection of Chikungunya virus E1 protein (CHIK-VP). Recombinant CHIK-VP has been taken here as an example to establish the sensing methodology. This new class of nanocomposite, containing fluorescent and an electrochemical sensing probe, can provide double responsive sensing of a single analyte to enhance the system's reliability over other liposome-based platforms. In addition to different concentrations of CHIK-VP, the specific antibody-conjugated QDs-liposome@MB and Fe 3 O 4 nanoparticles can bind with the CHIK-VP, and it can make a sandwich structure, as pictured in Scheme 1. The whole conjugates (QDs-liposome@MB with CHIK-VP-Fe 3 O 4 ) have been separated magnetically from the medium to discard the interferences and excess liposomes. A surfactant can then disrupt the liposomal formation and release the fluorophores and redox probes for analysis. Hence, several probes can come out from the conjugates in the presence of a few numbers of CHIK-VP, and the detection signal can be amplified in both processes of fluorescence and electrochemical differential pulse voltammetry (DPV), respectively. In combination with the magnetic separation and dual-mode detection, here, it can be expected to establish a new sensing mechanism where dual signals from a single analyte make more reliable testing results for real sample analysis. 

Dynamic light scattering (DLS) measurements were performed using a Zetasizer Nano series (Malvern Inst. Ltd., Malvern, UK). A confocal laser scanning microscope (FV-1000, Olympus, Tokyo, Japan) was used to take the liposome image using a stage thermocontrol system (Thermoplate, Tokai Hit, Shizuoka, Japan). Fluorescence spectra and UVvis absorption was taken by using a microplate reader (Infinite F500; TECAN, Ltd., Männedorf, Switzerland). Transmission electron microscopy (TEM) images of QDs, Fe 3 O 4 , liposomes, and their nanocomposites were obtained by JEOL TEM (JEOL, Tokyo, Japan). Electrochemical DPV was performed by an SP-150 (BioLogic.inc, Tokyo, Japan) in a saturated Ag/AgCl, with a conventional three-electrode cell consisting of a glassy carbon disk electrode (4 mm in diameter) as the counter, reference, and working electrodes, respectively (EC frontier, Tokyo, Japan).

Necessary precursors such as CdO, ODE, HDA, TOP, Se, and OA were used to perform the organometallic hot-injection synthesis of hydrophobic CdSe QDs followed by a previously reported procedure [32] .

The synthesis of magnetic Fe 3 O 4 nanoparticle was followed by a previously reported standard method [33, 34] . Assynthesized magnetic Fe 3 O 4 nanoparticles were coated with APTES by previously reported salinization method. To dissolve the APTES, dry toluene was used as the reaction medium, and finally, the as-synthesized Fe 3 O 4 nanoparticles were added into the solution. To obtain the APTES-coated Fe 3 O 4 nanoparticles, the mixture of the solution was refluxed at 120°C for 20 h with continuous stirring. Finally, the APTES-coated Fe 3 O 4 nanoparticles were rinsed with fresh toluene to remove the remaining APTES and were dried overnight and stored.

The as-synthesized hydrophobic CdSe QDs were centrifuged for 10 min at 11,000×g and then re-dispersed in chloroform to measure the concentration. Methylene blue (MB) solutions were prepared by dilution method from its stock solution of 10 mM in PBS.

Twenty microliters of hydrophobic CdSe QDs dissolved in chloroform and 200 μL of 10 mM phospholipid mixtures solution of DOPC: DOPG: DSPE-PEG 2000 (molar ratio 50:40:10) in chloroform were added into 5-mL glass vials and was evaporated by a flow of nitrogen gas to produce a thin homogeneous lipid film layer on the glass wall [22] . Then, the vial was stored in a vacuum desiccator for 12 h to evaporate completely. The fluorescence image of lipid film containing QDs is given in Fig. S1 , ESM. To make the homogeneous lipid suspension, 1 mL of the MB solution (various concentrations as mentioned later) was used to Liposome Scheme 1 Schematic representation of the formation of QDs-liposome@MB and its sandwich hybridization with Fe 3 O 4 nanoparticles and its dual-mode detection mechanism for CHIK-VP detection hydrate the lipid film and agitated on a vortex mixer until the lipid film was entirely detached from the glass walls. The process of forming the liposome has been schematically presented in Fig. S2 . Finally, the lipid suspension was dialyzed using a 2-kDa dialysis bag for 24 h to get purified unilamellar monodisperse QDs-liposome@MB by membrane filtering method.

The anti-CHIK-VP antibody was conjugated to the aminefunctionalized liposome and APTES-coated Fe 3 O 4 nanoparticles separately, according to the previously reported protocols [35] [36] [37] . Initially, the carboxyl group of antibodies was activated using EDC/NHS. After that, the as-synthesized QDs-liposome@MB and APTES-coated Fe 3 O 4 were added to conjugate the antibody separately and incubated for 1 h at room temperature. The amine group of DSPE-PEG 2000 phospholipids in the liposome and the APTES of Fe 3 O 4 nanoparticles conjugate with the activated carboxylic group of antibodies, and the solution was purified by centrifugation for 10 min at 10,000 rpm to remove the unreacted antibodies and the other coupling agents. The method of sensor preparation and its detection has been schematically presented in Fig. S2 in the ESM.

Antibody-conjugated QDs-liposome@MB and Fe 3 O 4 nanoparticles were added with various CHIK-VP concentrations, as mentioned later, and incubated for 10 min to make the sandwich structure. After the antibody-virus binding, an external magnet of 10 mT was placed at the bottom of the mixture solution to remove the detection solutions' impurities and excess reactants. After separating, the detection solution was re-dispersed in a fresh PBS buffer (pH 6.8) and transferred in a 96-well microplate. For the disruption of the liposome, 0.1 mM of 5 μL chloroform was added in the solution, as mentioned in earlier reports [14] , which triggered the liposome's disruption, releasing embedded QDs and encapsulated MB from the liposome. The solution was excited at 400 nm, and the fluorescence intensity was measured in a range of 630-750 nm before and after the addition of chloroform. Similarly, the solution was separately mixed with the PBS electrolyte. Electrochemical DPV was performed by an SP-150 (BioLogic.inc, Tokyo, Japan) in a conventional three-electrode cell consisting of a glassy carbon disk electrode (4 mm in diameter) as working, a Pt wire as counter and a saturated Ag/AgCl, electrode as a reference electrode (EC frontier, Tokyo, Japan) at a fix scan window of − 0.4 to 0.0 V.

To make the precise size of the liposome, the specific composition of phospholipids of DOPC, DOPG, and DSPE-PEG 2000 amine (50:30:20) were taken according to our previous reports [22] . As-synthesized, hydrophobic CdSe QDs were used to get embedded in the lipid bilayer of liposome during the film formation step of the lipid. MB was also incorporated while following the hydration step of the liposome so that MB can reside in the inner core of the liposome. After that, 0.2 μm of the polycarbonate membrane was used as a filter to remove the excess MB from the liposome solution. Simultaneously, Fe 3 O 4 nanoparticles were synthesized by the sol-gel method, and APTES was coated on it through the standard process of salinization. Initially, these three nanoparticles (QDs, Fe 3 O 4 , and liposome) were characterized by TEM, as shown in Fig. 1 .

In Fig. 1a , QDs are presented as uniformly dispersed with the range of 5-7 nm in size, where the particle distribution shows in Fig. 1b . The UV-absorption peak at 650 nm and the fluorescence spectra at 670 nm of the QDs indicate the successful preparation of the CdSe (Fig. 1c ) [38] . The QDs are dark red under UV light, as shown in Fig. 1d . The liposomes are shown in homogeneously distributed spherical form, as presented in the TEM image of Fig. 1e . The liposome structures are also characterized by the confocal images where the differential interference contrast (DIC) image (Fig. 1f ) and fluorescent image (Fig. 1g) 

The as-synthesized QDs-liposome@MB has been initially investigated for the suitable surfactant to release embedded fluorescent molecules. Among the different surfactants commonly used for the disruption of liposomal formation [39] , chloroform shows the best result compared to Triton X and Tween 20 (Fig. 2a) [40, 41] . In the case of a 1:1 mixture of chloroform and methanol, the liposome's initial perturbation is slightly higher than only chloroform. However, after some time of incubation, the release has been noticed highest in chloroform. As the dissolution of the lipid layer in the chloroform is a slow process, the optimized time for the complete release has been chosen as 10 min, as shown in Fig. 2b . After that, the fluorescence enhancement has reached its saturation, confirming the liposome's dissolution time is 10 min. The amount of CdSe QDs has also been optimized with the fixed amount of liposome of 10 3 particles mL −1 . Three different concentrations of QDs have been taken to check the optimum condition where the QDs can be entirely embedded in the liposome's hydrophobic surface. In the case of 0.5 and 1 mg mL −1 QD concentration, the fluorescence enhancement is quite satisfactory compared to the low level of 0.1 mg mL −1 . However, in the case of 1 mg mL −1 , the QDs are not only embedded in the surface but also penetrated inside the liposome, as shown in the inset of Fig. 2c . Due to the hydrophobic nature of the QDs, the encapsulated amount of the QDs inside the liposome is completely random and thus should be avoided. Therefore, the moderate concentration of 0.5 mg mL −1 of CdSe QDs has been selected for further liposome formation. A similar phenomenon is also observed in the case of DSPE lipid concentration. This amine groupcontaining lipid has been used in this work to produce the amine functionalization on the surface of the liposome, where the antibody can bind through its carboxylic group [42] . Therefore, it is always good to take the maximum amount of DSPE lipid in the lipid mixture composition without hampering the structure to load the maximum number of antibodies. However, a higher concentration than 1 mM can spill the QDs inside the core. Therefore, to avoid the QD encapsulation, less than 1 mM of the DSPE lipid has been used (Fig. 2d ).

In the electrochemical sensing, the concentration of the encapsulated redox probe is the most crucial parameter. The maximum concentration of MB can enhance sensitivity. However, the possibility of leakage or the background signal increases with the increasing concentration, resulting in reduced reliability [43] . Therefore, the encapsulation of MB concentration in the liposome was optimized. During the liposome synthesis, three levels of MB concentration (0.1, 1, and 5 mM) were used for the MB encapsulation in the core of the liposome and measured different concentrations of CHIK-VP. It is evident from Fig. 2e ; all different concentrations of MB display excellent linearity in the DPV signal. However, the sensor's blank value in the case of 5 mM concentration is very high, indicating the possible leakage of the MB. Therefore, the highest concentration of 5 mM MB has been rejected. Compared with 0.1 and 1 mM, peak intensities of 1 mM MB are best suited according to their slope of the calibration lines, which is used for the remaining studies.

Additionally, after optimizing different liposome composition with the concentration of the embedded QDs and encapsulated MB, the construction of the QDs-liposome@MB||CHIK-VP||Fe 3 O 4 nanoconjugates were investigated with a different concentration ratio of the Fe 3 O 4 nanoparticles and QDs-liposome@MB. In this sensing work, the analyte of CHIK-VP bound with antibody-conjugated QDs-liposome@MB and Fe 3 O 4 in a sandwich formation and then separated by applying a magnetic step. Therefore, it is obvious that the higher number of magnetic Fe 3 O 4 nanoparticles can increase magnetic separation efficiency. However, an excess amount of Fe 3 O 4 nanoparticles can bind on the virus surface itself rather than conjugate of the liposome, which may generate a false-negative signal. On the other side, the fewer amount of Fe 3 O 4 may be unable to bind the virus as well as with the liposomes, producing false-positive signals. Therefore, using the amount of magnetic Fe 3 O 4 nanoparticle should be optimized, which is also crucial in this work. A concentration range of 10 −13 -10 −8 g mL −1 of CHIK-VP has been applied to the different amounts of magnetic Fe 3 O 4 nanoparticles with a fixed concentration of QDs-liposome@MB. As shown in Fig. 2f , the magnetically isolated QDs-liposome@MB||CHIK-VP||Fe 3 O 4 nanoconjugates have been tested both fluorometric and DPV method before and after the addition of chloroform. In the case of a low amount of Fe 3 O 4 , the magnetic nanoconjugates contain a lesser amount of virus particle than the 0.7 mg, which indicates the partial binding of viruses.

On the other hand, a high amount of 1 mM (y=0.79x+12.5, 0.989)  1.0 mM (y=0.94x+14.6, 0.995)  5.0 mM (y=0.95x+15.1, 0.989 

After optimizing all the parameters, the QDs-liposome@MB-Fe 3 O 4 system was tested for the applicability for the detection of different concentrations of CHIK-VP. Before the addition of CHIK-VP, there are free antibody-conjugated QDs-liposome@MB and Fe 3 O 4 in the reaction medium. However, there is no substantial interaction between these two composites. After adding different concentrations of CHIK-VP and incubating for 10 min, the liposome and nanoparticles were bound with the virus through their corresponding specific antibodies conjugated on the surface of each to make the QDs-liposome@MB||CHIK-VP||Fe 3 O 4 sandwich structure. The nanoconjugates were separated magnetically and used to measure the virus concentration by the (Fig. 3b) , based on the intensity of fluorescence, conserves the linearity over the full range of concentration with a correlation coefficient of 0.977. The limit of detection (LOD) has been calculated and obtained from the calibration line, which is 0.56 pg mL −1 derived from the 3σ/s method (three times the standard deviation of the lowest concentration of target/slope of the calibration line) [44] . The TEM (Fig. 3c ) and confocal images ( Fig. 3d and e) of the nanoconjugates after chloroform addition also corroborate with our hypothesis about the disruption structure of the liposomes. Simultaneously, the QDs-liposome@MB||CHIK-VP||Fe 3 O 4 nanoconjugates have also been tested in the electrochemical DPV for the measurement of the released MB. Based on the hypothesis, with the addition of chloroform in the liposome mixture, the released MB comes into the buffer. As shown in Fig. 3f , the DPV signal of MB at − 0.22 V represents CHIK-VP concentration in the concentration range of 10 −14 -10 −8 g mL −1 . The calibration line accomplished from the peak current in DPV was calculated and plotted in Fig. 3g , which shows the linearity with the correlation coefficient of 0.993. The LOD has been measured from the calibration line of 32.7 fg mL −1 , calculated from the 3σ/s method [44] which is one order less than the optical detection due to the high sensitivity of the electrochemical process.

In comparison with other virus detection methods, as presented in Table 1 , the LOD and the range of concentration have clear superiority over other virus detection methods. As CHIK's detection or its protein is rarely reported in the literature, we have compared our results with other methods. In the case of colorimetric and fluorometric sensing, which are the most common detection method due to its simplicity and the possibility for naked-eye detection, the LOD is too poor, not applicable for CHIK diagnosis of this disease. In electrochemical methods, a relatively low LOD has been reported. However, due to the dual approach, the combination of fluorescent and electrochemical methods, the LOD, and the wide range of concentration range of this current work is significantly better than the others.

As the antibody conjugation controlled the sandwich structure formation between the target CHIK-VP with QDs-liposome@MB and Fe 3 O 4 nanoparticles, it is evident that the sensor should possess high specificity. To confirm its specificity, a selectivity test was performed with other viruses such as WSSV, Zika, influenza virus (10 5 copies mL −1 ), and hepatitis E virus-like particle (10 −9 g mL −1 ) and BSA as a negative control. The concentration of all interferences taken for selectivity is higher than their concentrations found in blood, serum, or other sources. The BSA does not show any significant signal in both the detection method of fluorometric and DPV (Fig. 4) as expected. Other interfering viruses do not significantly change the signal because the sensing method includes magnetic separation of impurities. A minimal fluorometric response has been observed, which may occur due to the nonspecific interaction with the liposome membrane, which is significantly low compared to the target CHIK-VP's signal. Therefore, from this selective study, it can be noted that the fluorescence and the DPV signal originates only if the specific target virus is present, which confirms its practical applicability for the virus detection purpose. However, for its real sample analysis, the stability of these materials is a major concern. Due to the formation of the liposomal platform, there is a possibility of leakage of the liposome structure's encapsulated materials over time. In Fig. S3 of ESM, the materials' stability shows acceptable results within 2 weeks of its preparation, a disadvantage for its practical analysis. However, the liposomal structure can be replaced by any other stable nanocarrier like solid-lipid nanoparticles or metalorganic frameworks to enhance the stability for the real sample analysis in the future.

In this work, a liposome-based dual-functional signal amplification system with the combination of magnetic Fe 3 O 4 nanoparticles has been developed to detect CHIK-VP. For the successful blending of these two components, few numbers of virus particles have been able to produce amplified intense signals even in presence of other interferences. A hydrophobic red fluorescent CdSe QDs embedded and MB solution encapsulated liposome with APTES-coated Fe 3 O 4 nanoparticles were prepared separately and conjugated to the anti-CHIK-VP antibody to make specific binding for the target virus. In presence of various CHIK-VP concentrations, the QDs-liposome@MB and magnetic Fe 3 O 4 nanoparticles formed the sandwich-like structured complex which was disrupted for the virus detection purpose. The LOD has been found as 0.56 pg mL −1 and 32.7 fg mL −1 in fluorometric and DPV process, respectively. Due to the successful fabrication of dual-mode detection probes in a single system, the liposomal matrix could be applied for double responsive sensing for a single analyte. This enhances the reliability of the results exceptionally well, signifying the proposed platform's superiority over other liposome-based systems. Also, the negligible cross-reactivity with other viruses and different matrices, along with low background signals, confirm the specific behavior of the sensor, indicating its potential application in different virus sensing approaches in the near future.

Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00604-020-04656-2. 

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