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© 2021 World Scientific Publishing Company

https://doi.org/10.1142/9789811227257_0001

Novel POC Bionanosensor for Direct Detection of RNA-Virus Infections in Minutes

May Maung^1,2,*, Chengde Cui^2,† and Saion Sinha^1,2,‡

¹ Biomedical Engineering Program, University of New Haven, West Haven, CT, USA

² 12-15 Molecular Diagnostics, Branford, CT, USA

^* mtzmaung007@gmail.com

^† chengde.cui@12-15MD.net

^‡ saion.sinha@12.15MD.com

Viral infections (like different types of influenza virus) are becoming alarmingly common often leading to epidemics. Since Viruses can multiply from genomic materials it can spread the infection quickly as well as cause other complications like birth defects, autoimmune disease and even cancer. Diagnostics of viral infection traditionally is performed using antigen-antibody reaction, which is time consuming and labor intensive. Also, viral infections can be latent or show false negative results when antibody reaction is tested. In-situ direct hybridization detection is possible by expensive, long and difficult radiation-based methods. Our group has developed a Carbon Nanotube (CNT) & Graphene based Bionanosensor (BNS) for rapid and direct detection of specific DNA sequences. In this technique, single-stranded (ss) DNA probe (known as priming) is immobilized on a substrate. This recognizes a specific complementary target DNA in a sample solution and gets hybridized leading to a detectable electrical signal. In this study we have expanded the scope of this BNS by using it to detect quick in-situ RNA hybridization. A sample of RNA was tested simultaneously in an array of 4 biosensors (each primed by the complementary sequence). The resistance across each of the biosensors was measured with a self-designed, feed-back controlled, balanced Wheatstone bridge circuit with an Arduino interface. A specific pattern related to hybridization was observed and a weighted average performed over 30 experiments. The sensors primed with the forward RNA was able to detect the complementary sequence in a mixed sample in 10 minutes from the starting time.

Keyword: biosensor POC; nanobiosensor; nanotechnology; RNA; virus diagnosis

Problem Statement: Virus are infectious micro-organisms which can only survive by invading a cell (called host), using it as a base, multiply very fast by spreading genomic material (primarily ribonucleic acid – RNA) and quickly build a colony on it. The quick growth of this viral colony is felt by the patient (whose host cells have been used) as the infection spreads, as well as cause other complications may develop like birth defects, autoimmune disease and even cancer. Diagnostics of viral infection are currently performed using antigen-antibody matching blood tests and cultures, which are time-consuming (2-3 days), labor intensive and prone to show false negative results. Sometimes there are no diagnostic tests available, particularly in the case when the virus mutates, which happens very frequently. Genetic/molecular tests, can avoid these issues but unfortunately cannot be performed directly by traditional methods as the RNA is very unstable. Thus prompt and accurate diagnostic procedures are necessary, as some viral infections have already become serious threat to the patient and public health the longer it takes to diagnose it.

Objective: In this paper, we demonstrate the development of a Carbon Nanotube (CNT) & Graphene based Bionanosensor (BNS) that performs rapid and direct detection of specific nucleic acid sequences-particularly RNA in a few minutes.

Methods: In this patented technique [1], single-stranded (ss) RNA probe is immobilized (known as priming) on a patented CNT-graphene substrate known as DTIM [2]. If the test-sample solution, contains the specific complimentary (ss-RNA) then it will hybridize with the RNA-probe leading to a detectable electrical signal. If the sample solution does not contain the specific complimentary ss-RNA, it will not hybridize (non-hybridization) and thus lead to a different but detectable electrical signal. In our experiment, 5 μL sample of RNA (Forward: 5’ CCG AUA CGC UGC CAA UCA GU 3’) was primed and its complementary sequence was tested simultaneously in an array of 4 biosensors at a time. The voltage and resistance across each biosensor were measured with a self-designed, feed-back controlled, balanced Wheatstone bridge circuit with an Arduino interface. This voltage/resistance data was averaged over several similar experiments in-situ with a Python Code. Since the hybridization is very temperature sensitive, the measurement was performed in a thermally insulated temperature controlled device. A specific pattern related to hybridization was observed and a weighted average performed for both hybridization and non-hybridization experiments. The sensors primed with the forward RNA were able to detect the complementary sequence in a mixed sample in 10 minutes from the starting time.

Results: When the complementary strand is added to the bionanosensor, two RNA strands bind on the surface of carbon nanotube resulting in an increase in the voltage (resistance) values due to decrease in conductivity .

On the other hand, when the non-complementary strand is added on the sensor, the two RNA strands cannot hybridize each other resulting in increase in conductivity due to binding of negatively charged RNA to CNT, thus leading to decrease in the voltage and resistance values.

Fig. 1. Total 9 hybridization experiments were averaged, and the average graph shows that the increasing pattern in Normalized voltage after complementary second drop of RNA.

Fig. 2. Total 9 non-hybridization experiments were averaged, and the average graph shows that the decreasing pattern in Normalized voltage after non-complementary second drop of RNA.

Conclusion: The results prove that the hybridization of RNA and non-hybridization of RNA sequences can be detected directly by using the patented CNT-graphene substrate within 2 minutes of applying the sample. This change in the resistance gradient between hybridized and non-hybridized experiments disappear after 2 minutes as the entropy of the process increases. Thus we have a biosensor capable of quick and direct diagnosis of viral infections.

References

1. US Patent # 9919922 “Bionanosensor Detection Device”

2. US Patent # 8623509 “Thermometric Carbon Composites”