(B) Spectrum shows SERS band assignment from WNV conjugated nanoassembly. probe was exhibited using another mosquito-borne chikungunya computer virus (CHIKV) as a negative control. Experimental data demonstrate a huge enhancement of SERS intensity is mainly due to the strong electric field enhancement, which has been confirmed by the finite-difference time-domain (FDTD) simulation. Reported FDTD simulation data indicate the SERS enhancement factor can be more than 104 occasions, due to the assembled structure. Reported results suggest that bio-conjugated AuNP-4G2 based SERS probes have great potential to be used to screen viral particles in clinical and research-based WNT-4 laboratories. Graphical Abstract Introduction According to the world health business (WHO)1C2, over one million people worldwide die from mosquito-borne diseases every year. Dengue computer virus (DENV) and West Nile computer virus (WNV) are the leading causative brokers of mosquito-borne diseases worldwide1C4. Aside from mosquito transmission, DENV and WNV are also transmitted by blood transfusions and organ transplantation1C4. Importantly, DENV has been identified as a high-priority infectious agent with the potential risk of transfusion-transmission in both the United States and Canada1C4. However, there is currently no routine screening of DENV in clinical settings, which is usually DCVC partially due to lack of a rapid, sensitive and cost-effective detection assay. Driven by the need, we report for the first time the development of an anti-flavivirus 4G2 antibody conjugated gold nanoparticle (AuNP-4G2)-based surface enhanced Raman spectroscopy (SERS) probe that can be used as a cost-effective and rapid detection tool for DENV and WNV selectively. SERS has the ability to rapidly detect microorganisms or biological analytes with chemical specificity intrinsic to vibrational spectroscopy5C14. Since the Raman signal can be enhanced by 108C1014 orders of magnitude in the presence of DCVC a metal nanomaterial surface15C23, SERS is usually emerging as an important tool for identification and classification of microorganisms24C30. Additionally, SERS has the ability to provide detailed information regarding the DCVC chemical composition of microorganisms and it can also serve as a fingerprint for detection and identification of microorganisms15C20. In addition to its fingerprinting ability and sensitivity, one of the other important features of the SERS assay is usually its specificity, which has been achieved here by attaching computer virus specific antibodies to the gold nanoparticle surface, as shown in Scheme 1. Open in a separate window Scheme 1 (A) Schematic representation of the construction of anti-flaviviral (4G2) coated gold nanoparticles. (B) Schematic representation shows detection of viruses using Raman fingerprinting. FDTD simulation data indicate a huge enhancement of the SERS intensity is due to strong electric field. Using the above advantages, we have developed an anti-flaviviral antibody coated AuNP-based SERS assay, as shown DCVC in Scheme 1, for rapid and sensitive detection of DENV and WNV selectively. Our reported results exhibited that antibody conjugated gold nanoparticles can be used as fingerprint spectra for viruses. Since the effective plasmon field generated by nanoparticle assemblies DCVC around the viral surface is usually more intense than individual nanoparticles, our reported experimental data exhibited that the detection limit is as low as 10 viruses/ml. Our experimental findings around the plasmon coupling enhanced SERS signal was supported by a finite-difference time-domain (FDTD) simulation26C30. FDTD is known to be a powerful tool for modeling electromagnetic near-field enhancement, which is an important parameter for enhancing SERS intensity via nanoparticle assembly. Since the SERS signal enhancement factor is usually approximately proportional to the fourth power of the electric field enhancement |and WNV gene31C32, and were purchased either by Integrated DNA Technologies (Coralville, IA) or Applied Biosystems (Grand Island, NY). All results were expressed as the absolute number of viral RNA copies/100 l of sample using the iTAQ? Universal Probes one-step qPCR kit (Bio-Rad, Hercules, CA) and were compared to viral gene standards for absolute copy number quantification. Results and Discussion To develop the selective SERS probe for WNV and DENV, anti-flaviviral antibodies (4G2) were conjugated to gold nanoparticles (AuNPs) with the detailed synthesis procedures described in methods. Physique 1A shows the image of the antibody attached-gold nanoparticles (AuNP-4G2), which indicates that this size is around 10C15 nm. The excitation spectrum, as shown in Physique 1D indicates that this plasmon band of AuNPs are very slightly shifted (2 nm) after conjugation with the antibody. Due to the lack of antigen-antibody interaction, gold nanoparticles do not form assembly structure in the absence of computer virus and as a result, we have not observed any broad absorption spectra from antibody conjugated gold nanoparticles. To determine whether AuNP-4G2 complexes bind to DENV and WNV, one thousand (103) plaque forming models (PFU) of viruses in PBS (50 l) were incubated for 15 minutes with the AuNP-4G2 complexes (500 l; 1:0.1 dilution), followed by fixation in 4% PFA. AuNP-4G2 probed with DENV-2 was then spun down (8,000.