Single Molecule Particle Analysis using Nanotechnology

Nanotechnology is the area of science that involves creation of devices/materials or systems in the nanometer scale. The last few decades have seen an increasing demand for rapid, sensitive, and cheaper diagnostic tools in healthcare. Advances in fabrication technologies have led to more miniaturized systems that are satisfying the promise of “micro total analysis” or “lab-on-chip” systems by facilitating the integration of multiple processing steps into a single device or multiple task-specific devices into a fluidic motherboard (i.e., modular microfluidics). The field of nanotechnology has the ability to revolutionize medical diagnostics by facilitating point-of-care testing with greater sensitivity even at the single molecule level. This allows for the screening of diseases at an early stage by identifying biomarkers of the diseases that are in extremely low concentrations in the blood (i.e., liquid biopsy). To this realization, we have used thermoplastics as our choice of material to fabricate microfluidic/nanofluidic hybrid systems that can evaluate how well a patient responds to chemotherapy, identify single nucleotide polymorphisms that cause major life threatening diseases such as stroke and caner, and development of nanofluidic devices to enumerate SARS CoV-2 viral particles that causes the novel coronavirus of 2019. We developed a high-throughput nanofluidic circuit on which single DNA molecules can be stretched to near their full contour length in nanochannels (<100 nm). Patients with cancer undergoing chemotherapy have more oxidative damage in their DNA compared to a healthy individual, which is an indicator of their response to therapy. We tested the device using calf thymus DNA standards labelled with a bis-intercalating dye and the abasic sites were labelled with another dye. Thus, the DNA molecules that were stretched in the nanochannels were parked and visualized using a fluorescent microscope. The abasic sites that were labelled were identified with their position in the DNA and the number of abasic sites per 105 nucleotides identified. This technique can be effectively used on samples having mass limits (picograms range) and where PCR cannot be utilized. Higher the number of abasic sites, better the response of the patient to chemotherapy, such as doxorubicin for breast cancer patients. While this nanofluidic circuit was used only to visualize the abnormalities in DNA, the next device we developed, called the nanosensor, facilitates the integration of multiple processes into a single device. The nanosensor was used to identify point mutations in DNA or mRNA responsible for diseases such as cancer and stroke, respectively. The device featured 8 pixel array populated with 1 µm pillars, which act as a solid support for Ligase Detection Reactions (spLDR) that can identify a single nucleotide mutation in a DNA from a large majority of wild type DNA. The spLDR can also identify mRNA transcripts from the design of spLDR primers that specifically recognize a unique transcript. The reaction is performed on the pixel arrays and the products are subsequently shuttled into nanometer flight tubes featuring two in-plane nanopores that act as resistive pulse sensors (RPS) to generate a current drop as the products pass through these pores. The time-of-flight (TOF) between the pores in series are used to distinguish between normal and mutated DNA, thus acting as a diagnostic appropriate for the precision medicine initiative. We were able to successfully fabricate the device, run COMSOL simulations to test operation using both hydrodynamic and electrokinetic flows, which were verified via experimentation to establish the functionality of the device to perform the above mentioned processes. The hydrodynamic flow operations used for spLDR was tested using Rhodamine B and the electrokinetic flow to inject the products of the spLDR into the flight tube was tested using oligonucleotides (25mer). Further, plastic-based nanofluidic devices were extended to detect the presence of SARS-CoV-2 viral particles using a nanopore of 350 nm in effective diameter, which has called a nano-coulter Counter (nCC). Briefly, saliva samples containing the viral particles were run through a microfluidic affinity chip containing pillars with surface-immobilized aptamers specific to the SARS-CoV-2 particles. The captured viral particles were released from the microfluidic chip using a blue light and the elute containing only the SARS viral particles were sent to the nCC, which used the RPS technique to count the number of particles. We designed multiple iterations of the nCC and used COMSOL simulations to guide device development. Using the combined principle of hydrodynamic and electrokinetic flow to introduce the viral particles into the nCC, we were able to detect patients with COVID-19 as well as estimate the viral load in SARS CoV-2 standards based on the frequency of the signals generated by correlating the results to a calibration curve. Thus, this combined multi-chip process can diagnose COVID-19 in <20 min thus venturing as an in-home diagnostic kit in the future by automating the operations into a hand-held device

Fluidic operation of a polymer-based nanosensor chip for analysing single molecules

Most medical diagnostic tests are expensive, involve slow turnaround times from centralized laboratories and require highly specialized equipment with seasoned technicians to carry out the assay. To facilitate realization of precision medicine at the point of care, we have developed a mixed-scale nanosensor chip featuring high surface area pillar arrays where solid-phase reactions can be performed to detect and identify nucleic acid targets found in diseased patients. Products formed can be identified and detected using a polymer nanofluidic channel. To guide delivery of this platform, we discuss the operation of various components of the device and simulations (COMSOL) used to guide the design by investigating parameters such as pillar array loading, and hydrodynamic and electrokinetic flows. The fabrication of the nanosensor is discussed, which was performed using a silicon (Si) master patterned with a combination of focused ion beam milling and photolithography with deep reactive ion etching. The mixed-scale patterns were transferred into a thermoplastic via thermal nanoimprint lithography, which facilitated fabrication of the nanosensor chip making it appropriate for in vitro diagnostics. The results from COMSOL were experimentally verified for hydrodynamic flow using Rhodamine B as a fluorescent tracer and electrokinetic flow using single fluorescently labelled oligonucleotides (single-stranded DNAs, ssDNAs)

Microfluidic Device for On-Chip Immunophenotyping and Cytogenetic Analysis of Rare Biological Cells

This work is licensed under a Creative Commons Attribution 4.0 International License.The role of circulating plasma cells (CPCs) and circulating leukemic cells (CLCs) as biomarkers for several blood cancers, such as multiple myeloma and leukemia, respectively, have recently been reported. These markers can be attractive due to the minimally invasive nature of their acquisition through a blood draw (i.e., liquid biopsy), negating the need for painful bone marrow biopsies. CPCs or CLCs can be used for cellular/molecular analyses as well, such as immunophenotyping or fluorescence in situ hybridization (FISH). FISH, which is typically carried out on slides involving complex workflows, becomes problematic when operating on CLCs or CPCs due to their relatively modest numbers. Here, we present a microfluidic device for characterizing CPCs and CLCs using immunofluorescence or FISH that have been enriched from peripheral blood using a different microfluidic device. The microfluidic possessed an array of cross-channels (2–4 µm in depth and width) that interconnected a series of input and output fluidic channels. Placing a cover plate over the device formed microtraps, the size of which was defined by the width and depth of the cross-channels. This microfluidic chip allowed for automation of immunofluorescence and FISH, requiring the use of small volumes of reagents, such as antibodies and probes, as compared to slide-based immunophenotyping and FISH. In addition, the device could secure FISH results in <4 h compared to 2–3 days for conventional FISH

In-plane Extended Nano-coulter Counter (XnCC) for the Label-free Electrical Detection of Biological Particles

This is the peer reviewed version of the following article: Z. Zhao, S. Vaidyanathan, P. Bhanja, S. Gamage, S. Saha, C. McKinney, J. Choi, S. Park, T. Pahattuge, H. Wijerathne, J. M. Jackson, M. L. Huppert, M. A. Witek, S. A. Soper, Electroanalysis 2022, 34, 1961., which has been published in final form at https://doi.org/10.1002/elan.202200091. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.We report an in-plane extended nanopore Coulter counter (XnCC) chip fabricated in a thermoplastic via imprinting. The fabrication of the sensor utilized both photolithography and focused ion beam milling to make the microfluidic network and the in-plane pore sensor, respectively, in Si from which UV resin stamps were generated followed by thermal imprinting to produce the final device in the appropriate plastic (cyclic olefin polymer, COP). As an example of the utility of this in-plane extended nanopore sensor, we enumerated SARS-CoV-2 viral particles (VPs) affinity-selected from saliva and extracellular vesicles (EVs) affinity-selected from plasma samples secured from mouse models exposed to different ionizing radiation doses

Microfluidic affinity selection of active SARS-CoV-2 virus particles

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus’s S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip’s surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent

Thermoplastic nanofluidic devices for identifying abasic sites in single DNA molecules

DNA damage can take many forms such as double-strand breaks and/or the formation of abasic (apurinic/apyrimidinic; AP) sites. The presence of AP sites can be used to determine therapeutic efficacy of many drugs, such as doxorubicin. While there are different assays to search for DNA damage, they are fraught with limitations, such as the need for large amounts of DNA secured from millions of cells. This is challenging due to the growing importance of using liquid biopsies as a source of biomarkers for many in vitro diagnostic assays. To accommodate the mass limits imposed by the use of liquid biopsies, we report a single-molecule DNA damage assay that uses plastic nanofluidic chips to stretch DNA to near its full contour length when the channel dimensions (width and depth) are near the persistence length (∼50 nm) of double-stranded (ds) DNA. The nanofluidic chip consisted of input funnels for high loading efficiency of single DNA molecules, entropic traps to store the DNA and simultaneously load a series of nanochannels for high throughput processing, and an array of stretching nanochannels to read the AP sites. Single dsDNA molecules, which were labeled with an intercalating dye and a biotinylated aldehyde reactive probe (bARP), could be parked in the stretching nanochannels, where the AP sites were read directly using a dual-color fluorescence microscope equipped with an EMCCD camera. One color of the microscope was used to read the DNA length and the second color detected the AP sites. The nanofluidic chip was made from thermoplastics via nanoimprint lithography, which obviated the need for direct writing the devices in glass or quartz using focused ion beam milling. We show that we can read the frequency of AP sites in single dsDNA molecules with the frequency of AP sites determined by associating fluorescently-labeled streptavidin with bARP through a biotin/streptavidin complex

Tailoring Thermoplastic In-Plane Nanopore Size by Thermal Fusion Bonding for the Analysis of Single Molecules

We report a simple method for tailoring the size of in-plane nanopores fabricated in thermoplastics for single-molecule sensing. The in-plane pores were fabricated nanoimprint lithography (NIL) from resin stamps, which were generated from Si masters. We could reduce the size of the in-plane nanopores from 30 to ∼10 nm during the thermal fusion bonding (TFB) step, which places a cover plate over the imprinted polymer substrate under a controlled pressure and temperature to form the relevant nanofluidic devices. Increased pressures during TFB caused the cross-sectional area of the in-plane pore to be reduced. The in-plane nanopores prepared with different TFB pressures were utilized to detect single-λ-DNA molecules resistive pulse sensing, which showed a higher current amplitude in devices bonded at higher pressures. Using this method, we also show the ability to tune the pore size to detect single-stranded (ss) RNA molecules and single ribonucleotide adenosine monophosphate (rAMP). However, due to the small size of the pores required for detection of the ssRNA and rAMPs, the surface charge arising from carboxylate groups generated during O plasma oxidation of the surfaces of the nanopores to make them wettable had to be reduced to allow translocation of coions. This was accomplished using EDC/NHS coupling chemistry and ethanolamine. This simple modification chemistry increased the event frequency from ∼1 s to \u3e136 s for an ssRNA concentration of 100 nM

Microfluidic affinity selection of active SARS-CoV-2 virus particles

We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus\u27s S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip\u27s surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent