Detection of airborne viruses using electro-aerodynamic deposition and a field-effect transistor.
ABSTRACT: We report a technique for the detection of aerosolized viruses. Conventional field-effect-transistor (FET)-based techniques use solution-based processes, thus require antibody binding to the detection region of the FET prior to the supply of the analyte. With the method described here, virus-antibody-bound particles are delivered to the FET during detection; therefore, neither a pre-treatment antibody binding step on the FET channel nor washing process for virus-antibody-binding are necessary. Our method is based on the concept that virus-antibody-bound particles are larger than the virus or antibody alone, and thus have larger charge numbers following aerosol charging. When these particles are charged by negative ions and electro-aerodynamically deposited on a substrate, there exists a location on the substrate where neither lone virus nor antibody particles land, and where only virus-antibody-bound particles are deposited. If this location coincides with the channel of the FET, the resulting variation in the current can be used to indicate the existence of a virus. By aerosolizing a mixed solution of the virus and the antibody, only the virus-antibody-bound particles were transported to the swCNT-FET, and the electric current in the swCNT-FET decreased to 30% of that measured with no deposited particles.
Project description:Biosensors employing single-walled carbon nanotube field-effect transistors (SWCNT FETs) offer ultimate sensitivity. However, besides the sensitivity, a high selectivity is critically important to distinguish the true signal from interference signals in a non-controlled environment. This work presents the first demonstration of the successful integration of a novel peptide aptamer with a liquid-gated SWCNT FET to achieve highly sensitive and specific detection of Cathepsin E (CatE), a useful prognostic biomarker for cancer diagnosis. Novel peptide aptamers that specifically recognize CatE are engineered by systemic in vitro evolution. The SWCNTs were firstly grown using the thermal chemical vapor deposition (CVD) method and then were employed as a channel to fabricate a SWCNT FET device. Next, the SWCNTs were functionalized by noncovalent immobilization of the peptide aptamer using 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker. The resulting FET sensors exhibited a high selectivity (no response to bovine serum albumin and cathepsin K) and label-free detection of CatE at unprecedentedly low concentrations in both phosphate-buffered saline (2.3 pM) and human serum (0.23?nM). Our results highlight the use of peptide aptamer-modified SWCNT FET sensors as a promising platform for near-patient testing and point-of-care testing applications.
Project description:A single-walled carbon nanotube (SWCNT) in a field-effect transistor (FET) configuration provides an ideal electronic path for label-free detection of nucleic acid hybridization. The simultaneous influence of more than one response mechanism in hybridization detection causes a variation in electrical parameters such as conductance, transconductance, threshold voltage and hysteresis gap. The channel length (L) dependence of each of these parameters necessitates the need to include them when interpreting the effect of L on the response to hybridization. Using the definitions of intrinsic effective mobility (µe) and device field-effect mobility (µf), two new parameters were defined to interpret the effect of L on the FET response to hybridization. Our results indicate that FETs with ?300 µm long SWCNT exhibited the most appreciable response to hybridization, which complied with the variation trend in response to the newly defined parameters.
Project description:Label-free and real-time detection technologies can dramatically reduce the time and cost of pharmaceutical testing and development. However, to reach their full promise, these technologies need to be adaptable to high-throughput automation. To demonstrate the potential of single-walled carbon nanotube field-effect transistors (SWCNT-FETs) for high-throughput peptide-based assays, we have designed circuits arranged in an 8 × 12 (96-well) format that are accessible to standard multichannel pipettors. We performed epitope mapping of two HIV-1 gp160 antibodies using an overlapping gp160 15-mer peptide library coated onto nonfunctionalized SWCNTs. The 15-mer peptides did not require a linker to adhere to the non-functionalized SWCNTs, and binding data was obtained in real time for all 96 circuits. Despite some sequence differences in the HIV strains used to generate these antibodies and the overlapping peptide library, respectively, our results using these antibodies are in good agreement with known data, indicating that peptides immobilized onto SWCNT are accessible and that linear epitope mapping can be performed in minutes using SWCNT-FET.
Project description:Detection of tumor markers is important for cancer diagnosis. Field-effect transistors (FETs) are a promising method for the label-free detection of trace amounts of biomolecules. However, detection of electrically charged proteins using antibody-immobilized FETs is limited by ionic screening by the large probe molecules adsorbed to the transistor gate surface, reducing sensor responsiveness. Here, we investigated the effect of probe molecule size on the detection of a tumor marker, α-fetoprotein (AFP) using a FET biosensor. We demonstrated that the small receptor antigen binding fragment (Fab), immobilized on a sensing surface as small as 2-3 nm, offers a higher degree of sensitivity and a wider concentration range (100 pg/mL-1 μg/mL) for the FET detection of AFP in buffer solution, compared to the whole antibody. Therefore, the use of a small Fab probe molecule instead of a whole antibody is shown to be effective for improving the sensitivity of AFP detection in FET biosensors. Furthermore, we also demonstrated that a Fab-immobilized FET subjected to a blocking treatment, to avoid non-specific interactions, could sensitively and selectively detect AFP in human serum.
Project description:Tobacco mosaic virus (TMV) has been employed as a robust functional template for the fabrication of a TMV/zinc oxide field effect transistor (FET). A microwave based approach, under mild conditions was employed to synthesize stable zinc oxide (ZnO) nanoparticles, employing a molecular precursor. Insightful studies of the decomposition of the precursor were done using NMR spectroscopy and material characterization of the hybrid material derived from the decomposition was achieved using dynamic light scattering (DLS), transmission electron microscopy (TEM), grazing incidence X-ray diffractometry (GI-XRD) and atomic force microscopy (AFM). TEM and DLS data confirm the formation of crystalline ZnO nanoparticles tethered on top of the virus template. GI-XRD investigations exhibit an orientated nature of the deposited ZnO film along the c-axis. FET devices fabricated using the zinc oxide mineralized virus template material demonstrates an operational transistor performance which was achieved without any high-temperature post-processing steps. Moreover, a further improvement in FET performance was observed by adjusting an optimal layer thickness of the deposited ZnO on top of the TMV. Such a bio-inorganic nanocomposite semiconductor material accessible using a mild and straightforward microwave processing technique could open up new future avenues within the field of bio-electronics.
Project description:Preventing exposure of virulent pathogens during aerosolizing procedures such as intubations has been a cause of concern during the coronavirus pandemic. As such, protocols have been adjusted and precautions implemented in order to minimize the risk to the proceduralist. As patients improve, we face another high-risk aerosolizing procedure-extubation. We illustrate a protocol to help minimize the exposure risk during extubation. We describe a barrier technique during extubation which contained aerosolized particulates into a non-rebreather mask at time of extubation. Our protocol allows providers to perform extubations while minimizing exposure to aerosolized particles.
Project description:The performance of a norovirus antigen detection assay was assessed using monoclonal antibody NV23 and single-chain antibody HJT-R3-A9 to identify both virus-like particles and virus-containing fecal samples. The detection of 25 different norovirus genotypes as recombinant virus-like particles or in clinical samples was dependent on virus or antigen concentration.
Project description:We demonstrate a new protein detection methodology based upon frequency domain electrical measurement using silicon nanowire field-effect transistor (SiNW FET) biosensors. The power spectral density of voltage from a current-biased SiNW FET shows 1/f-dependence in frequency domain for measurements of antibody functionalized SiNW devices in buffer solution or in the presence of protein not specific to the antibody receptor. In the presence of protein (antigen) recognized specifically by the antibody-functionalized SiNW FET, the frequency spectrum exhibits a Lorentzian shape with a characteristic frequency of several kilohertz. Frequency and conventional time domain measurements carried out with the same device as a function of antigen concentration show more than 10-fold increase in detection sensitivity in the frequency domain data. These concentration-dependent results together with studies of antibody receptor density effect further address possible origins of the Lorentzian frequency spectrum. Our results show that frequency domain measurements can be used as a complementary approach to conventional time domain measurements for ultrasensitive electrical detection of proteins and other biomolecules using nanoscale FETs.
Project description:Rapid, sensitive, and direct label-free capture and characterization of nanoparticles from complex media such as blood or serum will broadly impact medicine and the life sciences. We demonstrate identification of virus particles in complex samples for replication-competent wild-type vesicular stomatitis virus (VSV), defective VSV, and Ebola- and Marburg-pseudotyped VSV with high sensitivity and specificity. Size discrimination of the imaged nanoparticles (virions) allows differentiation between modified viruses having different genome lengths and facilitates a reduction in the counting of nonspecifically bound particles to achieve a limit-of-detection (LOD) of 5 × 10(3) pfu/mL for the Ebola and Marburg VSV pseudotypes. We demonstrate the simultaneous detection of multiple viruses in a single sample (composed of serum or whole blood) for screening applications and uncompromised detection capabilities in samples contaminated with high levels of bacteria. By employing affinity-based capture, size discrimination, and a "digital" detection scheme to count single virus particles, we show that a robust and sensitive virus/nanoparticle sensing assay can be established for targets in complex samples. The nanoparticle microscopy system is termed the Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and is capable of high-throughput and rapid sizing of large numbers of biological nanoparticles on an antibody microarray for research and diagnostic applications.
Project description:Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 103 PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.