Development of disposable electrode for the detection of mosquito-borne viruses.

Development of disposable, rapid, and convenient biosensor with high sensitivity and reliability is the most desired method of viral disease prevention. To achieve this goal, in this work, a practical impedimetric biosensor has been implemented into a disposable electrode on a screen-printed carbon electrode (SPCE) for the detection of two mosquito-borne viruses. The biosensor fabrication has step-wisely carried out on the disposable electrode surface at room temperature: starting from conductive film formation, physical binding of the gold nanoparticles (AuNPs)-polyaniline (PAni) into the conductive film, and biofunctionalization. To get the maximum efficiency of the antibody, biotinylated antibody has been conjugated on the surface of AuNP-PAni/PAni-SPCE via the streptavidin-biotin conjugation method which is a critical factor for the high sensitivity. Using the antibody-antigen interaction, this disposable electrode has designed to detect mosquito-borne infectious viruses, Chikungunya virus (CHIKV), and Zika virus (ZIKV) separately in a wide linear range of 100 fg mL-1 to 1 ng mL-1 with a low detection limit of 1.33 and 12.31 fg mL-1 , respectively.

Improvement of Modular Protein Display Efficiency in SpyTag-Implemented Norovirus-like Particles.

Genetic fusion and chemical conjugation are the most common approaches for displaying a foreign protein on the surface of virus-like particles (VLPs); however, these methods may negatively affect the formation and stability of VLPs. Here, we aimed to develop a modular display platform for protein decoration on norovirus-like particles (NoV-LPs) by combining the NoV-LP scaffold with the SpyTag/SpyCatcher bioconjugation system, as the NoV-LP is an attractive protein nanoparticle to carry foreign proteins for various applications. The SpyTagged-NoV-LPs were prepared by introducing SpyTag peptide into the C-terminus of the norovirus VP1 protein. To increase surface exposure of the SpyTag peptide on the NoV-LPs, two or three repeated extension linkers (EAAAK) were inserted between the SpyTag peptide and VP1 protein. Fluorescence proteins, EGFP and mCherry, were fused to SpyCatcher and employed as SpyTag conjugation partners. These VP1-SpyTag variants and SpyCatcher-fused EGFP and mCherry were separately expressed in silkworm fat bodies and purified. This study reveals that adding an extension linker did not disrupt the VLP formation; instead, it increased the particle size by 4-6 nm. The conjugation efficiency of the VP1-SpyTag variants with the extended linker improved from ∼15-35 to ∼50-63% based on the densitometric analysis, while it was up to 77% based on an optical quantification of EGFP and mCherry. Results indicate that the linker causes the SpyTag peptides to be positioned further away from the C-termini of VP1 and potentially increases the exposure of the SpyTag to the outer surface of the NoV-LPs, allowing more SpyTag/SpyCatcher complex formation on the VLP surface. Our study provides a strategy for enhancing the conjugation efficiency of NoV-LP and demonstrates the platform's utility for developing vaccines or functional nanoparticles.

Advancement of dengue virus NS1 protein detection by 3D-nanoassembly complex gold nanoparticles utilizing competitive sandwich aptamer on disposable electrode.

Signal amplification have been centralized in developing the highly reliable biosensor for analyte detection with a narrow detection window. We proposed an aptasensor to provide a highly sensitive early-stage diagnostic platform of dengue virus NS1 protein (DENV-NS1) by dual-approach - colorimetric and electrochemical detection. This work utilized two different aptamers specific to DENV-NS1: One conjugated to gold nanoparticles (AuNPs), forming AuNPs-Apt1 and its complementary sequence aptamer, forming AuNPs-Apt2. The unbound Apt1 of AuNPs-Apt1 by DENV-NS1 were to hybridize to AuNPs-Apt2 and induced a 3D-nanoassembled formation, resulting in DENV-NS1 concentration-dependent plasmonic color change. Occurrence of the hybridization of Apt1 and Apt2, the 3D-assembled hybridized aptamers of AuNPs was incubated with methylene blue (MB) solution, which intercalated a high number of MB molecules within the duplex structure of aptamers, and the complex was captured on the Apt2-conjugated disposable gold electrode (DGE). The developed aptamer-based biosensor showed high sensitivity with colorimetric response down to 1.28 pg/mL and electrochemical approach down to 30 fg/mL of DENV-NS1 with good selectivity. This work showcases an advanced utilization of aptamer and its complementary anti-sense aptamer in signal amplification and nanocarrier for biosensing.

Design and Analysis of a Single System of Impedimetric Biosensors for the Detection of Mosquito-Borne Viruses.

The treatment for mosquito-borne viral diseases such as dengue virus (DENV), zika virus (ZIKV), and chikungunya virus (CHIKV) has become difficult due to delayed diagnosis processes. In addition, sharing the same transmission media and similar symptoms at the early stage of infection of these diseases has become more critical for early diagnosis. To overcome this, a common platform that can identify the virus with high sensitivity and selectivity, even for the different serotypes, is in high demand. In this study, we have attempted an electrochemical impedimetric method to detect the ZIKV, DENV, and CHIKV using their corresponding antibody-conjugated sensor electrodes. The significance of this method is emphasized on the fabrication of a common matrix of gold-polyaniline and sulfur, nitrogen-doped graphene quantum dot nanocomposites (Au-PAni-N,S-GQDs), which have a strong impedimetric response based only on the conjugated antibody, resulting in minimum cross-reactivity for the detection of various mosquito-borne viruses, separately. As a result, four serotypes of DENV and ZIKV, and CHIKV have been detected successfully with an LOD of femtogram mL-1.

Sulfur-doped carbon dots@polydopamine-functionalized magnetic silver nanocubes for dual-modality detection of norovirus.

Synergistic dual-mode optical platforms are up-and-coming detection tools in the diagnosis and management of infectious diseases. Here, novel dual-modality fluorescence (FL) and surface-enhanced Raman scattering (SERS) techniques have been integrated into a single probe for the rapid and ultrasensitive detection of norovirus (NoV). The developed FL-SER-based biosensor relies on the dual-signal enhancements of newly synthesized sulfur-doped agar-derived carbon dots (S-agCDs). The antigen-antibody immunoreaction results in forming a core-satellite immunocomplex between anti-NoV antibody-conjugated S-agCDs and polydopamine-functionalized magnetic silver nanocubes [poly (dop)-MNPs-Ag NCs]. By deploying an immunomagnetic enrichment protocol and performing the SERS modality on a single-layer graphene substrate, norovirus-like particles (NoV-LPs) were detected across a wide range of 1 fg mL-1 - 10 ng mL-1 with an excellent limit of detection of 0.1 fg mL-1. The combined advantage of the dual-signaling properties of the biosensor was demonstrated using FL confocal imaging for "hotspots" tracking prior to SERS detection of clinical NoV in fecal specimen down to ⁓10 RNA copies mL-1. The proposed dual-modality biosensor's performance increases the prospect of a rapid and low-cost sensitive NoV detection and surveillance option for public health.

Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

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 Fe3O4 nanoparticles (Fe3O4 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. Graphical abstract.

Controlling distance, size and concentration of nanoconjugates for optimized LSPR based biosensors.

In this report, we have examined the distance- and size-dependent localized surface plasmon resonance (LSPR) between fluorescent quantum dots (QDs) and adjacent gold nanoparticles (AuNPs) to provide a comprehensive evaluation, aiming for practical application in biosensing platform. A series of peptides with different chain lengths, connected between QDs and AuNPs is initially applied to prepare various CdSe QDs-peptide-AuNP systems to optimize LSPR signal. Separation distance between two nanoparticles of these systems before and after conjugation is also confirmed by quantum mechanical modeling and corroborated with their LSPR influenced fluorescence variations. After detailed optimizations, it can be noted that larger sized AuNPs make strong quenching of QDs, which gradually shows enhancement of fluorescence with the increment of distance and the smaller sized AuNPs. Depending on the requirement, it is possible to tune the optimized structure of the CdSe QD-peptide-AuNP nanostructures for the application. In this work, two different structural designs with different peptide chain length are chosen to construct two biosensor systems, observing their fluorescence enhancement and quenching effects, respectively. Using different structural orientation of these biosensors, two nanoconjugates has applied for detection of norovirus and influenza virus, respectively to confirm their application in sensing.

Fluorometric virus detection platform using quantum dots-gold nanocomposites optimizing the linker length variation.

In this study, a tunable biosensor using the localized surface plasmon resonance (LSPR), controlling the distance between fluorescent CdZnSeS/ZnSeS quantum dots (QDs) and gold nanoparticles (AuNPs) has been developed for the detection of virus. The distance between the AuNPs and QDs has been controlled by a linkage with a peptide chain of 18 amino acids. In the optimized condition, the fluorescent properties of the QDs have been enhanced due to the surface plasmon effect of the adjacent AuNPs. Successive virus binding on the peptide chain induces steric hindrance on the LSPR behavior and the fluorescence of QDs has been quenched. After analyzing all the possible aspect of the CdZnSeS/ZnSeS QD-peptide-AuNP nanocomposites, we have detected different concentration of influenza virus in a linear range of 10-14 to 10-9 g mL-1 with detection limit of 17.02 fg mL-1. On the basis of the obtained results, this proposed biosensor can be a good alternative for the detection of infectious viruses in the various range of sensing application.

Dual modality sensor using liposome-based signal amplification technique for ultrasensitive norovirus detection.

Sensitive and accurate detection methods for infectious viruses are the pressing need for effective disease diagnosis and treatment. Herein, based on V2O5 nanoparticles-encapsulated liposomes (VONP-LPs) we demonstrate a dual-modality sensing platform for ultrasensitive detection of the virus. The sensing performance relies on intrinsic peroxidase and electrochemical redox property of V2O5 nanoparticles (V2O5 NPs). The target-specific antibody-conjugated VONP-LPs and magnetic nanoparticles (MNPs) enrich the virus by magnetic separation and the separated VONP-LPs bound viruses are hydrolyzed to release the encapsulated V2O5 NPs. These released nanoparticles from captured liposomes act as peroxidase mimics and electrochemical redox indicator resulting in noticeable colorimetric and robust electrochemical dual-signal. Utilizing the superiority of dual-modality sensor with two quantitative analysis forms, norovirus like particles (NoV-LPs) can be detected by electrochemical signals with a wide linear range and low detection limit. To verify the applicability in real samples, norovirus (NoV) collected from actual clinical samples are effectively-identified with excellent accuracy. This proposed detection method can be a promising next-generation bioassay platform for early-stage diagnosis of virus disease and surveillance for public health.

Use of Target-Specific Liposome and Magnetic Nanoparticle Conjugation for the Amplified Detection of Norovirus.

Viral diseases are one of the most life-threatening diseases as they can erupt unpredictably and spread rapidly in any medium with a very small number of particles. Therefore, the key for lethal virus detection should be highly sensitive in the early-stage detection, which can help increase the chance of survival. Amplification of the detecting signal is one of the most promising mechanisms for the detection of low-concentration analytes. A proper amplification can develop such a kind of system where a small number of particles can produce intense signals for a prominent detection. Keeping this in mind, in this report, we have presented a fluorometric method to detect norovirus (NoV) by a newly developed fluorophore-labeled liposome and a magnetically modified Fe3O4 combined system. Homogeneously distributed amine-functionalized liposomes have been constructed filled with a strong fluorophore of calcein. Simultaneously, (3-aminopropyl)-triethoxysilane (APTES)-functionalized Fe3O4 nanoparticles are also synthesized by the standard silanization process, and these two separately synthesized nanoparticles were functionalized with an antibody to achieve specificity. The Fe3O4 and calcein-liposome system has been applied for NoV detection, which was magnetically separated from the analyte medium and then externally burst to release the fluorophores from the core of the liposome. The easiness, rapidity, and sensitivity in a wide linear range can offer a huge potential of this method in point-of-care diagnostics.

The detection and identification of dengue virus serotypes with quantum dot and AuNP regulated localized surface plasmon resonance.

The dengue hemorrhagic fever or dengue shock syndrome has become a severe human fatal disease caused by infection with one of the four closely related but serologically distinct dengue viruses (DENVs). All four dengue serotypes are currently co-circulating throughout the subtropics and tropics. Since the fatality rate increases severely when a secondary infection occurs by a virus serotype different from that of the initial infection, serotype identification is equally important as virus detection. In this study, the development and validation of a rapid and quantitative DENV serotype-specific (serotypes 1-4) biosensor are reported by optimizing the stable system between cadmium selenide tellurium sulphide fluorescent quantum dots (CdSeTeS QDs) and gold nanoparticles (AuNPs). Four different nanoprobes are designed using each primer-probe serotype-specific hairpin single-stranded DNA covalently bound at different positions to CdSeTeS QDs, which generates an altered fluorescence signal for each serotype of DENV. In fourplex reactions with free functionalized AuNPs and the four nanoprobes, the standard dilutions of the target virus DNA from 10-15 to 10-10 M were successfully detected. The limit of detection was found to be in the femtomolar range for all four serotypes, where the serotype detection ability was undoubtedly established. To confirm the applicability of this sensing performance in long chained complex RNAs, the sensor was also applied successfully to RNAs extracted from DENV culture fluids for serotype identification as well as quantification, which can lead to a potential diagnostic probe for point-of-care detection.

Femtomolar Detection of Dengue Virus DNA with Serotype Identification Ability.

Dengue surveillance trusts only on reverse transcription-polymerase chain reaction (RT-PCR) type methodologies for confirmation of dengue virus serotypes; however, its real time application is restricted due to the expensive, complicated, and time-consuming process. In search of a new sensing system, here, we have reported a two-way-detection method for Dengue virus (DENV) serotype identification along with DNA quantification by using a new class of nanocomposite of gold nanoparticles (AuNP) and nitrogen, sulfur codoped graphene quantum dots (N,S-GQDs). The N,S-GQDs@AuNP has been used for serotype detection via a simple fluorescence technique using four dye-combined probe DNAs which is further validated by confocal microscopy. The quantification of the DNA has been measured by the differential pulse voltammetric (DPV) technique using methyelene blue as a redox indicator. Results obtained in this study, clearly demonstrate that the N,S-GQDs@AuNP can efficiently detect the four serotypes of DENV individually in the concentration range of 10-14 to 10-6 M with the LOD of 9.4 fM. In addition, to confirm its applicability in long chained complex DNA system, the sensor was also applied to the clinically isolated DENV DNA and showed satisfactory performances for serotype identification as well as quantification. We hope this simple and reliable method can pave an avenue for the development of sensitive and robust sensing probes in biomedical applications.

Single-step detection of norovirus tuning localized surface plasmon resonance-induced optical signal between gold nanoparticles and quantum dots.

A new method of label free sensing approach with superior selectivity and sensitivity towards virlabel-freeon is presented here, employing the localized surface plasmon resonance (LSPR) behavior of gold nanoparticles (AuNPs) and fluorescent CdSeTeS quantum dots (QDs). Inorganic quaternary alloyed CdSeTeS QDs were capped with L-cysteine via a ligand exchange reaction. Alternatively, citrate stabilized AuNPs were functionalized with 11-mercaptoundecanoic acid to generate carboxylic group on the gold surface. The carboxylic group on the AuNPs was subjected to bind covalently with the amine group of L-cysteine capped CdSeTeS QDs to form CdSeTeS QDs/AuNPs nanocomposites. The fluorescence of CdSeTeS QDs/AuNPs nanocomposite shows quenched spectrum of CdSeTeS QDs at 640 nm due to the close interaction with AuNPs. However, after successive addition of norovirus-like particles (NoV-LPs), steric hindrance-induced LSPR signal from the adjacent AuNPs triggered the fluorescence enhancement of QDs in proportion to the concentration of the target NoV-LPs. A linear range of 10-14 to 10-9 g mL-1 NoV-LPs with a detection limit of 12.1 × 10-15 g mL-1 was obtained. This method was further applied on clinically isolated norovirus detection, in the range of 102-105 copies mL-1 with a detection limit of 95.0 copies mL-1, which is 100-fold higher than commercial ELISA kit. The superiority of the proposed sensor over other conventional sensors is found in its ultrasensitive detectability at low virus concentration even in clinically isolated samples. This proposed detection method can pave an avenue for the development of high performance and robust sensing probes for detection of virus in biomedical applications.