In-Situ Monitoring of Real-Time Loop-Mediated Isothermal Amplification with QCM: Detecting Listeria monocytogenes

Abstract

Functionalized DNA sequences are promising sensing elements to combine with transducers for bio-sensing specific target microbes. As an application example, this paper demonstrates in situ detection of loop-mediated isothermal amplification products by hybridizing them with thiolated-ssDNA covalently anchored on the electrodes of a quartz crystal microbalance (QCM). Such hybridization leads to a frequency signal, which is suitable for monitoring real-time LAMP amplification based on mass-sensing: it detects interactions between the complementary nucleobases of LAMP products in solution and the thiolated-ssDNA probe sequence on the gold surface. Target DNA LAMP products cause irreversible frequency shifts on the QCM surfaces during hybridization in the kHz range, which result from both changes in mass and charge on the electrode surface. In order to confirm the LAMP assay working in the QCM sensing system at elevated temperature, the sky blue of positive LAMP products solution was achieved by using the Hydroxy Naphthol Blue (HNB) and agarose gel electrophoresis. Since on-QCM sensing of DNA hybridization leads to irreversible sensor responses, this work shows characterization by X-ray photoelectron spectroscopy (XPS) core spectra of S2p, N1s, Mg1s, P2p and C1s. XPS results confirmed that indeed both DNA and by-products of LAMP attached to the surface. Listeria monocytogenes DNA served to study in-situ detection of amplified LAMP products on DNA-functionalized surfaces.

Label-free and portable field-effect sensor for monitoring RT-LAMP products to detect SARS-CoV-2 in wastewater

“…Thus, this experiment was successful to determine that the detection of RT-LAMP products could be finished in less than 30 min. This was an interesting finding owing that, by using RT-LAMP, it reduces the testing time because the isothermal amplification has a fast response [ 47 ]; and, hence, 30 min are enough to complete the reaction with a readable signal.…”

Precision Temperature Control System with Low EMI for Applications in Analyzing Thermal Properties of Highly Sensitive Piezoelectric Sensors

“…QCM-based measurements often take tens of minutes to complete [ 4 , 6 ], hence the proposed system was tested for the long-term stability. Three setpoint values were chosen: 25 °C which is very often used for electronic components testing and characterization, and highest and lowest stable temperatures achieved, i.e., −3 °C and 70 °C.…”

“…This allows for external factors to impact the resonant frequency and damping factor of the exposed crystal, which makes it possible to indirectly measure various physical quantities. QCM properties make it suitable for many applications in modern science such as biosensors [ 4 , 5 , 6 , 7 ], vapors and particulate matter sensors [ 8 , 9 , 10 ], systems for characterization of liquids [ 11 , 12 , 13 ], and aerospace [ 14 , 15 , 16 , 17 , 18 ]. Underlying most experiments are indirect measurements of mass changes and viscoelastic properties [ 19 ].…”

“…There is a lot of recent research in this field in which QCMs are used in wide temperature ranges. One example is the listeria monocytogenes detection method developed by Wachiralurpan, S. et al In this study, QCM was used as the DNA hybridization biosensor operating at 60 °C [ 6 ]. Another example is research on low-temperature adsorption and fouling of milk fractions on stainless steel conducted by Holly A. Huellemeier et al in 25, 50, and 65 °C [ 22 ].…”

“…This is equal to a 324 Hz frequency drop, which according to Equation (1) corresponds to the 1.12 µg mass increase at the QCM surface, which has to be compensated. In Wachiralurpan, S. et al paper measurements were also performed at 60 °C [ 6 ]; therefore temperature effects have to be compensated but also the temperature stability has to be taken into account. According to the user manual of Thermo Haake ® DC30, the thermostat used during discussed experiments has a temperature stability of ±10 mK and accuracy of ±100 mK, which for measured mass change of 300 ng adds up to uncertainty of ±4.95 ng.…”