Rapid molecular diagnostics at home is the future of preventive medicine

(Nanowerk Highlights) Advanced molecular diagnostic tests that can quickly and precisely detect disease in the home could revolutionize preventive medicine and improve global biosecurity. Nucleic acid amplification tests (NAAT) such as polymerase chain reaction (PCR) provide high sensitivity and specificity for the identification of infectious diseases by amplifying the DNA and RNA of pathogens.

However, centralized laboratory-based NAAT requires complex and tedious sample processing steps such as pathogen lysis, nucleic acid purification and enzymatic amplification. These manual procedures rely on trained personnel operating expensive equipment, limiting accessibility. Conventional laboratory protocol of spin column based nucleic acid extraction for molecular diagnostics for COVID-19. Step 1: sample collection and transportation. Types of biological specimens are collected by various collection procedures, such as nasopharyngeal swab, nasal swab, and oropharyngeal swab, and transported to a centralized laboratory as soon as possible after collection. Of particular interest for molecular epidemiological analysis is where samples can be collected most easily and effectively at the lowest cost. Step 2: heat deactivation. Viruses are inactivated by incubating clinical samples at high temperatures to destabilize viral proteins and assemblies, rendering them incapable of infecting during downstream manual operations. Step 3: lysis/digestion. The lysis buffer contains high concentrations of salts and chaotropic detergents. Chaotropes disrupt hydrogen interactions and cause destabilization of proteins and nucleases. Organic amphipathic detergents break down the structure of cell membranes by dissociating membrane proteins with detergent hydrophobic moieties from the membrane. Proteases can be included in the lysis buffer to digest protein contaminants and degrade nucleases. The lysis buffer shows higher efficiency at high temperatures. Step 4: binding. The lysate is transported to the spin column. The chaotropic salt provides favorable conditions for the transfer of nucleic acids to the spin column silica membrane by creating a hydrophobic environment to break down the association between the NAs and water. Meanwhile, the chaotrope provides positively charged cations to saturate the silica membrane, thereby enhancing the uptake of the negatively charged NAs phosphate backbone under hydrophobic conditions. Since NAs is insoluble in ethanol, addition of ethanol will increase the precipitation of nucleic acids onto the silica membrane. Step 5: Washing the washing buffer, with ethanol as the predominant component, removes impurities such as protein polysaccharide residues. Several washing steps are usually performed to thoroughly remove residual contamination and solute buffers. Ethanol residue should be avoided after the washing step because it can prevent elution of sequential NAs and inhibit nucleic acid amplification. Step 6: elution. Elution buffer or purified water at pH 8-9 is usually used to release the NAs from the silica membrane to the bottom of the centrifuge tube during centrifugation. Step 7: reverse transcription-polymerase chain reaction (RT-PCR). The purified RNA was reverse transcribed into complementary DNA (cDNA), followed by cDNA amplification and fluorescence detection. (Reprinted with permission by Wiley-VCH Verlag)

An integrated microfluidic platform that combines automated sample preparation with on-chip NAAT in one miniature device offers a practical solution for performing precise molecular diagnostics at home without a dedicated laboratory.

Reviews on Advanced Materials (“Towards Fast and Accurate Molecular Diagnostics at Home”) evaluate recent advances in integrated sample preparation for NAAT using common clinical samples such as blood, urine, saliva, and faeces.

Blood provides many genetic and infectious disease biomarkers. However, invasive sampling via venipuncture or fingerprick is required to obtain blood. The integrated microcapillary loop-mediated isothermal amplification (LAMP) method successfully extracts DNA from a small 200 nL blood sample obtained with a minimally invasive finger prick, avoiding the need for expensive and painful traditional blood draws.

Urine offers a completely noninvasive liquid biopsy, in contrast to blood. However, urine contains much lower levels of nucleic acid biomarkers. Therefore, a microfluidic magnetic bead platform is required to capture and concentrate rare pathogenic DNA/RNA from large volumes of urine prior to extraction. Concentration increases the detection limit.

Saliva and nasal swabs allow non-invasive sampling of respiratory infections by means of viral particles secreted in mucus. The integrated microfluidic cartridge extracts HIV ribonucleic acid (RNA) biomarkers from as little as 30 μL of saliva in just 10 minutes, demonstrating the potential for extremely fast and sensitive sample-to-answer NAAT.

Stools contain gastrointestinal pathogens and can noninvasively identify lower gastrointestinal infections. However, viscous faecal samples require mechanical homogenization of the chip prior to pathogen capture and nucleic acid extraction.

Recently, an ingenious sharp-end acoustofluidic system actively homogenizes the feces inside the micro-ducts by generating strong flow forces with ultrasonically oscillating microscopic structures fabricated from silicon.

The integrated reusable microfluidic chip flexibly processes a wide variety of clinical sample types on a common platform:

The microfluidic channel is packed with silica microbeads which extract purified RNA from raw cellular lysates prior to on-chip detection of nucleic acid sequence (NASBA) based amplification without cross-contamination between samples.

The self-integrated blood analysis chip successfully integrates plasma separation from small 5–10 µL blood specimens with a downstream multiplex immunoassay that detects protein biomarkers.

The modular microfluidic platform allows for interchangeable single-use testing cartridges customized for specific sample types such as sputum or blood, providing a versatile tool for a wide range of nucleic acid and protein diagnostic assays. Integrated on-chip sample preparation that can be reused with a wide range of biological samples. a) Integrated microfluidic RNA purification room and real-time NASBA device. 1: Sample load; 2: buffer washing; 3: RNA elution; 4: denaturation of secondary and tertiary RNA structures; 5: NASBA enzyme load; 6: amplification process. b) Self-contained integrated microfluidic blood analysis system. Blood is pushed into the microchannels by degas-driven flow, and plasma is separated by trapping red and white blood cells in an integral moat structure. c) Microfluidic biomolecular amplification (µBAR) readers, including microfluidic cartridges and monitors. d) On-chip valve-assisted microfluidic chip demonstrates automation of reagent control. Mechanical sliders control the downward movement of the on-chip valves to open and close the channels, and the reagents flow through the desired chambers in sequence. e) Universal microfluidic cartridge for the diagnosis of M. tuberculosis (MTB) from sputum samples. f) i-STAT microfluidic cartridge consisting of a thin film electrode sensor on a silicon chip for target detection. Airbag suppression by intake motor (Reprinted with permission by Wiley-VCH Verlag) (click on image to enlarge)

On-chip NAAT offers many advantages over conventional benchtop laboratory testing methods: Portability, automation and speed – The thin film microfabrication heater integrated within the microfluidic chip enables fast thermal cycling to support on-chip PCR DNA amplification. Bubble prevention – Ingenious bubble-free microfluidic PCR chip prevents the formation of vapor bubbles using a watertight polyethylene vapor barrier and vacuum ducts integrated on the chip to remove bubbles. Bubbles disrupt the nanoliter reaction volume. Photonic heating – The patterned gold film deposited on the chip enables precise photonic heating by converting light to heat efficiently to achieve fast thermal cycles without contact with the complex heating block. Digital microfluidic partition – Microfluidic digital PCR improves analytical sensitivity by partitioning small samples into thousands of 50–250 nL wells to detect single DNA molecules, greatly simplifying integration with upstream sample preparation compared to complex laboratory quantitative PCR instruments.

In-house NAAT paired with automated integrated microfluidic sample preparation could enable frequent population screening during viral outbreaks, providing invaluable epidemiological data to stop disease in its tracks. Their comfort and accessibility in low resource settings is critical to improving preventive medicine on a global scale. In the not too distant future, wearable flexible microfluidic biosensors could provide on-site molecular diagnostic capabilities using easily accessible microliter volumes of body fluids such as sweat or saliva.

In short, the innovative lab-free microfluidic NAAT platform brings the powerful capabilities of advanced nucleic acid testing right into the home, workplace, or even the pocket of the average consumer to provide on-demand decentralized diagnostics that can help stop outbreaks before they happen. to spread.

By

Michael
Berger

– Michael is the author of three books by the Royal Society of Chemistry:
Nano-Society: Pushing the Boundaries of Technology,
Nanotechnology: A Small Future And
Nanoengineering: Skills and Tools for Making Technology Invisible
Copyright ©




Nanowerk LLC