Versatile nanorobot hand biosensor for specific capture and ultrasensitive quantification of viral nanoparticles

Abstract

Accurate determination of the concentration and viability of the viral vaccine vectors is urgently needed for preventing the spread of the viral infections, but also supporting the development and assessment of recombinant virus-vectored vaccines. Herein, we describe a nanoplasmonic biosensor with nanoscale robot hand structure (Nano RHB) for the rapid, direct, and specific capture and quantification of adenovirus particles. The nanorobot allows simple operation in practical applications, such as real-time monitoring of vaccine quantity and quality, and evaluation of vaccine viability. Modification of the Nano RHB with branched gold nanostructures allow rapid and efficient assessment of human adenovirus viability, with ultrahigh detection sensitivity of only 100 copies/mL through one-step sandwich method. Nano RHB detection results were consistent with those from the gold standard median tissue culture infectious dose and real-time polymerase chain reaction assays. Additionally, the Nano RHB platform showed high detection specificity for different types of viral vectors and pseudoviruses. Altogether, these results demonstrate that the Nano RHB platform is a promising tool for efficient and ultrasensitive assessment of vaccines and gene delivery vectors.

Keywords: Biosensor; Label-free detection; Nanoparticles; Surface plasmon resonance; Virus vector.

Graphical abstract

Fig. 1

One-step rapid quantification of viral particles using the Nano RHB platform. a) NanoSPR sensor modified by highly sensitive nanorobot hands. b) Scheme of the NanoSPR sensor immobilized receptor protein or antibody to form a NanoRHB platform with specific capture ability. c) Schematics of the minute-time scale of the Nano RHB platform for detecting viral vectors within 5 ​min. d) Schematics of the ultra-sensitivity scale of the Nano RHB platform for detecting viral vectors using one-step sandwich method within 15 ​min.

Fig. 2

Characteristics of the NanoSPR chip and AuNPs. a) Photograph of 12-inch Ti–Ag–Au NanoSPR chip. b) Integration of the Ti–Ag–Au NanoSPR chip with a standard 96-well plate. c) Integration of the NanoSPR chip with two 16-well plates. d) Photograph of a 16-well chip plate and microscope images of NanoSPR chip with two drops of PBST and sucrose solution on the chip surface. e–g) Scanning electron microscope images of repeating nanocup array with 200 ​nm diameter and f) 450 ​nm height, and of g) AuNPs with 30 ​nm diameter.

Fig. 3

a) Microscopic images and SEM images of the NanoSPR chip modified with L-cysteine and chloroauric acid at 0.2–1.5 ​mM. b) Measurement of the absorption spectral changes of the NanoSPR chip in water and in 5% sucrose after the chip was modified with a range of dopamine and chloroauric acid concentrations (0.2–1.5 ​mM). c) Relative OD changes at 590 and 570 ​nm after surface modification of the sensor chip. Data are shown as mean ​± ​SD (n ​= ​3). The statistical comparison between groups was performed following the student's t-test (two-tailed). ∗p ​< ​0.01, ∗∗p ​< ​0.001 and ∗∗∗p ​< ​0.0001. d,e) Atomic force microscopy image of the (d) unmodified NanoSPR chip and (e) NanoSPR chip under the optimal modification condition.

Fig. 4

Label-free detection of adenovirus using a CAR-coated Nano RHB platform within 5 ​min. a) Schematic diagram of adenovirus recognition based on the CAR-coated Nano RHB platform. b) Absorption differential spectra at 500–700 ​nm using the CAR-coated Nano RHB assay for adenovirus detection in the concentration ranges of 2.5 ​× ​10⁴ to 1.6 ​× ​10⁶ TCID₅₀/mL. c) Standard curve of the CAR-coated Nano RHB for adenovirus detection (R² ​= ​0.996). d) Kinetic binding curves of adenovirus at the OD difference values (OD₅₉₀−OD₅₇₀) based on the CAR-coated Nano RHB, with a detection range of 2.5 ​× ​10⁴ to 1.6 ​× ​10⁶ TCID₅₀/mL. e) Stability of the Nano RHB at different storage temperatures and time periods were tested using three standard positive samples. Data are shown as mean ​± ​SD (n ​= ​3).

Fig. 5

Label-free detection of adenovirus using the FX-modified NanoSPR platform. a) Schematic of adenovirus captured by the FX-coated Nano RHB. b) Differential spectra at 500–700 ​nm of the FX-coated Nano RHB for adenovirus detection with different concentrations (2.5 ​× ​10⁴–1.6 ​× ​10⁶ TCID₅₀/mL). c) Two-wavelength OD differential value change of the Nano RHB at 570 and 590 ​nm regarding different adenovirus concentrations (R² ​= ​0.990). d) Kinetic binding curves of the FX-coated Nano RHB at the OD difference values (OD₅₉₀−OD₅₇₀)for adenovirus detection with different concentrations (2.5 ​× ​10⁴–1.6 ​× ​10⁶ TCID₅₀/mL). e) Comparison of CAR- and FX-modified Nano RHB platform assays and adenovirus titers assay for six adenovirus samples. Data are shown as mean ​± ​SD (n ​= ​3). f) Fluorescence and bright field images of transfected cells at day 9 post-transfection.

Fig. 6

Label-free detection of total adenovirus with AuNP-enhanced nanoplasmonic virus capture platform. a) Schematic diagram of the nanoplasmonic virus capture platform integrated with AuNPs for detection of adenoviruses. b) Absorption spectrum of adenovirus at 500–700 ​nm, with a detection range of 1 ​× ​10²–1.6 ​× ​10⁶ copies/mL. c) Standard curve for adenovirus (R² ​= ​0.985). d) Kinetic binding curve of adenovirus determined at the OD difference values (OD₆₀₀−OD₅₈₀) using an anti-adenovirus antibody-coated microarray, with a detection range of 1 ​× ​10²–1.6 ​× ​10⁶copies/mL. e) Stability of the Nano RHB platform integrated with AuNPs at different storage temperatures and time periods using three standard positive samples. Data are shown as mean ​± ​SD (n ​= ​3).

Fig. 7

Comparison of the detection outcome for adenovirus of the Nano RHB platform with that of other detection methods. a) Comparison of qPCR assay and Nano RHB platform to quantify the concentration of adenovirus. Data are shown as mean ​± ​SD (n ​= ​3). The statistical comparison between groups was performed following the student's t-test (two-tailed). ∗p ​< ​0.01, and ∗∗p ​< ​0.001. b) Curve fitting of the qPCR and Nano RHB assays for adenovirus quantification detection. c) Specificity of the Nano RHB platform for the detection adenovirus as compared with four other pseudoviruses and four viral vectors.