【Scientific research trends】Professor Liu Gang team of Huazhong University of Science and Technology: Rapid quantitative detection of gene vaccine vectors black technology

On October 1, 2022, the team of Professor Liu Gang of Huazhong University of Science and Technology published a research paper
titled "Versatile nanorobot hand biosensor for speci?c capture and ultrasensitive quanti?cation of viral nanoparticles" in the journal Materials Today Bio.

During the COVID-19 pandemic, adenoviruses have become an ideal vaccine vector
to prevent infection, serious illness and death.
The entry of the vector into the body induces an immune response and builds immune memory requires an infectious active viral vaccine
.
In traditional quantitative detection methods, whether it is qPCR detection with high sensitivity or traditional immunological principle Elisa detection, only the total concentration
of viral vectors can be quantified.
Due to the interference of viral vector fragments, it is impossible to accurately determine the titer concentration of viral vaccine vectors, which in turn affects the quality control of vaccine production and the precise administration of vaccines
.
To prevent the spread of viral infections, there is an urgent need to accurately determine the concentration and viability of viral vaccine vectors and to support the development and evaluation
of recombinant viral vector vaccines.

In response to the above problems, the team of Professor Liu Gang and Dr.
Huang Liping of Huazhong University of Science and Technology, together with Professor Jin Meilin of Huazhong Agricultural University and Professor Zhang Qiang's team, developed a nano-plasma (Nano RHB) biosensor with nano-scale multi-functional robotic hand structure for rapid, non-destructive and specific capture and quantitative detection of adenoviral particles
.
The sensor can be used to monitor the quantity and quality of viral vaccine vectors in real time and assess the infection viability of viral vaccine vectors
.

In the study, the researchers first developed a label-free NanoSPR biosensor
based on the Very Light Transmission (EOT) effect.
This biosensor can be excited by unpolarized light and does not require complex optical equipment
.
Then, by directly implementing the gold seed growth method on the nanocup array chip, a new multifunctional NanoSPR biosensor
with nanorobot hands was designed.
Due to its special nanocup structure as a seed template, branched gold nanostructures
of different shapes are generated on the surface of the chip by polydopamine molecules.
These branched gold nanostructures function similarly to intelligent robotic hands, enhancing the SPR resonance effect to improve the sensitivity of the chip while increasing the surface area to improve the chip modification efficiency
.
Integrating these ultrasensitive biosensors into standard 96-well or 32-well plates allows direct monitoring of dynamic binding curves and quantitative detection of adenoviral vectors or vaccines using trace samples
.

The results show that, based on this novel sensor, recombinant human CAR, FX protein or antiadenoviral hexao-neighbor protein antibodies are fixed on the surface of NanoRHB, and these ligands can bind adenoviral fibrin (protruding spurs on the viral capsid) and hexaoliton proteins (the main capsid proteins), respectively, and can quickly detect adenovirus survival viability and concentration through adenovirus-specific binding CAR/FX protein interactions, detecting up to 96 samples simultaneously and at high throughput in 5 minutes.
More efficient and faster
than traditional viral titer detection methods and PCR methods.

In addition, the researchers also used lyophilized technology to integrate CAR-labeled gold particles into a monoclonal antibody-modified Nano RHB platform, and the detection sensitivity can be further improved by 5 quilts
.
The results of the study show that the unique SPR effect of the gold particle-coupled Nano RHB enables one-step sandwich for highly sensitive and rapid adenoviral vector particle evaluation without the need for washing steps and complex sample pretreatment
.
This ultra-sensitive gold nano-branched biochip can not only quickly and effectively assess the viability of adenoviral vectors, but also improve detection sensitivity to 100 copies/mL
by one-step sandwich method.

The researchers will further use the NanoRHB platform to examine 26 cell supernatant samples, and the results show that the results are very consistent
with the results of traditional qPCR and viral titer detection assays.
Nano RHB sensor analysis significantly reduces the total detection time, from days and hours to minutes, and improves detection capability
by directly binding to viral surface proteins to simultaneously quickly detect viral viability and concentration.
The new detection platform also demonstrates high specificity for different types of viral vectors
and pseudoviruses.
The results show that the Nano RHB platform is a promising high-throughput bioassay tool for efficient and ultra-sensitive evaluation of vaccines and gene delivery vectors, for rapid quality control of large-scale adenoviral vector vaccines, and for other viral vector- and gene vector-based assays
.

The research team has translated the above technology to develop an adeno-associated virus (AAV-2) quantitative detection kit based on NanoSPR technology, which can accurately, quickly, easily and high-throughput detect the AAV-2 virus content in cell supernatant
.

Li Rui, Ph.
D.
student of Huazhong University of Science and Technology, Zhao Ya, doctoral student of Huazhong Agricultural University, and Fan Hongli, doctoral student of Huazhong University of Science and Technology, are the co-first authors of the paper, and Dr.
Huang Liping and Professor Liu Gang of Huazhong University of Science and Technology, and Proof Jin Meilin and Professor Zhang Qiang of Huazhong Agricultural University are co-corresponding authors
of the paper.
The work has been funded by the National Key Research and Development Program of the Ministry of Science and Technology and the National Natural Science Foundation of China
.

Paper Link: https://doi.
org/10.
1016/j.
mtbio.
2022.
100444