Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples.

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.

Optimized acoustic biochip integrated with microfluidics for biomarkers detection in molecular diagnostics.

The development of integrated platforms incorporating an acoustic device as the detection element requires addressing simultaneously several challenges of technological and scientific nature. The present work was focused on the design of a microfluidic module, which, combined with a dual or array type Love wave acoustic chip could be applied to biomedical applications and molecular diagnostics. Based on a systematic study we optimized the mechanics of the flow cell attachment and the sealing material so that fluidic interfacing/encapsulation would impose minimal losses to the acoustic wave. We have also investigated combinations of operating frequencies with waveguide materials and thicknesses for maximum sensitivity during the detection of protein and DNA biomarkers. Within our investigations neutravidin was used as a model protein biomarker and unpurified PCR amplified Salmonella DNA as the model genetic target. Our results clearly indicate the need for experimental verification of the optimum engineering and analytical parameters, in order to develop commercially viable systems for integrated analysis. The good reproducibility of the signal together with the ability of the array biochip to detect multiple samples hold promise for the future use of the integrated system in a Lab-on-a-Chip platform for application to molecular diagnostics.

Plasma nanotextured polymeric lab-on-a-chip for highly efficient bacteria capture and lysis.

We describe the design, fabrication, and successful demonstration of a sample preparation module comprising bacteria cell capture and thermal lysis on-chip with potential applications in food sample pathogen analysis. Plasma nanotexturing of the polymeric substrate allows increase of the surface area of the chip and the antibody binding capacity. Three different anti-Salmonella antibodies were directly and covalently linked to plasma treated chips without any additional linker chemistry or other treatment. Then, the Ab-modified chips were tested for their capacity to bind bacteria in the concentration range of 10(2)-10(8) cells per mL; the module exhibited 100% efficiency in Salmonella enterica serovar Typhimurium bacteria capture for cell suspensions below 10(5) cells per mL (10(4) cells injected with a 100 µL sample volume) and efficiency higher than 50% for 10(7) cells per mL. Moreover, thermal lysis achieved on-chip from as low as 10 captured cells was demonstrated and shown to compare well with off-chip lysis. Excellent selectivity (over 1 : 300) was obtained in a sample containing, in addition to S. Typhimurium and E. coli bacteria.

Acoustic detection of DNA conformation in genetic assays combined with PCR.

Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost.

Sensitivity of the acoustic waveguide biosensor to protein binding as a function of the waveguide properties.

The aim of this work is to study the effect of operating frequency, piezoelectric substrate and waveguide layer thickness on the sensitivity of the acoustic waveguide sensor during the specific binding of an antibody by a protein. Shear horizontal (SH) wave devices consisting of (a) a LiTaO3 substrate operating at 104 MHz, (b) a quartz substrate operating at 108 MHz and (c) a quartz substrate operating at 155 MHz were coated with a photoresist polymer layer in order to produce acoustic waveguide devices supporting a Love wave. The effect of the thickness of the polymer layer on the Love wave was assessed by measuring the amplitude and phase of the wave before and after coating. The sensitivity of the above three biosensors was compared during the detection of the specific binding of different concentrations of Immunoglobulin G in the range of 0.7-667 nM to a protein A modified surface. Results indicate that the thickness of the polymer guiding layer is critical for obtaining the maximum sensitivity for a given geometry but a trade-off has to be made between the theoretically determined optimum thickness for waveguiding and the device insertion loss. It was also found that increasing the frequency of operation results in a further increase in the device sensitivity to protein detection.

Comparative study of IgG binding to proteins G and A: nonequilibrium kinetic and binding constant determination with the acoustic waveguide device.

The aim of this work was to measure and compare the binding constants of antibody immunoglobulin G (IgG) to bacterial cell wall proteins, streptococcal protein G and Staphylococcus aureus protein A, using an acoustic wave sensor. Devices, which used shear-horizontal acoustic waves propagating in a waveguide configuration at 108 and 155 MHz, were employed in the detection of apparent IgG binding constants at the solid-liquid interface in the range of 6.7-667 nM IgG. Real-time data during IgG-protein G and IgG-protein A binding yielded apparent association constants of 3.29 x 10(4) and 8.02 x 10(3) M(-1) s(-1) leading to equilibrium constants of 1.13 x 10(8) and 2.90 x 10(7) M(-1), respectively. The measured apparent rate constants are consistent with literature reports of higher affinity of protein G for IgG. Furthermore, protein binding through the Fc region of IgG is suggested to occur below 333 nM, while different mechanisms are suggested to occur above 333 nM. For the first time, nonequilibrium studies of IgG-protein G and A binding at a solid-liquid interface has yielded valuable quantitative kinetic information about binding mechanisms. The promise of this detection method is shown by providing quick determination of binding constants with low sample volumes.

Pulse mode operation of Love wave devices for biosensing applications.

In this work we present a novel pulse mode Love wave biosensor that monitors both changes in amplitude and phase. A series of concentrations of 3350 molecular weight poly(ethylene glycol) (PEG) solutions are used as a calibration sequence for the pulse mode system using a network analyzer and high frequency oscilloscope. The operation of the pulse mode system is then compared to the continuous wave network analyzer by showing a sequence of deposition and removal of a model mass layer of palmitoyl-oleoyl-sn-glycerophosphocholine (POPC) vesicles. This experimental apparatus has the potential for making many hundreds of measurements a minute and so allowing the dynamics of fast interactions to be observed.

Study of the sensitivity of the acoustic waveguide sensor.

The sensitivity of the acoustic waveguide sensor to mass deposition in the presence of liquid was optimized as a function of the over-layer thickness. The waveguide geometry consisted of a 0.2-2.2-microm poly(methyl)methacrylate (PMMA) over-layer deposited on the surface of a shear acoustic wave device and supported a Love wave. The response of each polymer-coated waveguide was initially assessed by monitoring the frequency and insertion loss of the device in the presence of air. Sensitivity to viscous and mass loading was studied by recording the amplitude and phase of the wave during the application of water and of a supported lipid bilayer, respectively, on the device surface. Supported bilayers are a versatile system for mass calibration in the presence of liquid because they can be formed spontaneously on a hydrophilic surface, resulting in a layer of reproducible mass density. Results clearly showed that the response of both amplitude and phase depends on the over-layer thickness and increases with the thickness of the polymer layer. Phase was generally found to be more sensitive than amplitude to both viscous water and mass loading. The maximum sensitivity to vesicles deposition was measured at 250 cm2 g(-1) and was detected when 1.3 microm of PMMA was used as a waveguide layer. Results showed that the sensitivity of the acoustic wave sensor can be improved by simply increasing the thickness of the PMMA and that supported phospholipid layers can form an ideal system for both mass calibration and interfacial modification.

Antibody binding to a functionalized supported lipid layer: a direct acoustic immunosensor.

A direct immunosensor has been developed using an acoustic wave device as a transducer. The device is based on an acoustic waveguide geometry that supports a Love wave. The biorecognition surface, formed on a gold layer, consisted of a biotinylated supported lipid layer which specifically bound streptavidin and, subsequently, biotinylated goat IgG. The modified surface was used as a model immunosensor and successfully detected rabbit anti-goat IgG in the concentration range 3 x 10(-8) - 10(-6) M. Using the anti-goat IgG binding isotherm and the time-resolved measurements of antibody binding, both the binding and rate constants of the reaction were determined. The specificity of each binding step was studied with the acoustic wave device, and it was concluded that the phospholipid bilayer showed a good suppression of nonspecific binding. Comparative measurements using surface plasmon resonance allowed the response of the immunosensor to be quantitatively correlated with mass binding to the surface.

Immunosensors.

Immunosensors are important analytical tools for monitoring antibody-antigen reactions in real time. Recent developments in immunosensors have produced systems that allow rapid and continuous analysis of the binding event without the requirement for added reagents or separation/washing steps. As a result, great interest has focused on commercializing immunosensors for applications in areas such as clinical, environmental and food analysis.

A novel Love-plate acoustic sensor utilizing polymer overlayers.

A Love-plate sensor, consisting of a surface skimming bulk wave (SSBW) device coated with a polymer layer, was found to increase the acoustic signal through coupling of the SSBW wave to a Love wave. Insertion loss, phase and frequency measurements were used to assess the optimum thickness of the polymer layer and the sensitivity of the device to mass-loading and viscous coupling.