Kelvin probe force microscopy (KPFM) allows the detection of single binding events between immunoglobulins (IgM, IgG) and their cognate antibodies (anti-IgM, anti-IgG). Here an insight into the reliability and robustness of the methodology is provided. Our method is based on imaging the surface potential shift occurring on a dense layer of ∼5 × 107 antibodies physisorbed on a 50 μm × 90 μm area when assayed with increasing concentrations of antigens in phosphate buffer saline (PBS) standard solutions, in air and at a fixed scanning location. A comprehensive investigation of the influence of the main experimental parameters that may interfere with the outcomes of KPFM immune-assay is provided, showing the robustness and reliability of our approach. The data are supported also by a thorough polarization modulation infrared reflection–absorption spectroscopy (PM-IRRAS) analysis of the physisorbed biolayer, in the spectral region of the amide I, amide II and amide A bands. Our findings demonstrate that a 10 min incubation in 500 μL PBS encompassing ≈ 30 antigens (100 zM) triggers an extended surface potential shift that involves the whole investigated area. Such a shift quickly saturates at increasing ligand concentration, showing that the developed sensing platform works as an OFF/ON detector, capable of assessing the presence of a few specific biomarkers in a given assay volume. The reliability of the developed methodology KPFM is an important asset in single molecule detections at a wide electrode interface.
Herein, we report an ultrasensitive and highly selective analytical methods to detect 2,4-dichlorophenoxyacetic acid (2,4-D) using an o-phenylenediamine based molecularly imprinted polymer (o-PD-MIP) sensor. Electrochemical Quartz Crystal Microbalance (EQCM) is used to investigate both the kinetics and the mass of the electropolymerized o-phenylenediamine. Additionally, successful removal of the template from the imprinted cavities is confirmed by comparing the XP spectra of imprinted and o-PD-MIPs after various template removal procedures. The most effective method involves a 70:30 mixture of MeOH:H2O for 15 min under stirring. The o-PD-MIP sensor exhibits high sensitivity with a LoD of (3 ± 1) × 10−12 M, which is below the EU regulation limits for drinking water by six orders of magnitude, a linear range between 10 and 100pM, and an excellent selectivity. These results proved the effectiveness of template removal procedure by using a 70:30 MeOH:H2O mixture and are a proof-of-concept for ultrasensitive and selective 2,4-D detection in real samples.
Screening asymptomatic organisms (humans, animals, plants) with a high-diagnostic accuracy using point-of-care-testing (POCT) technologies, though still visionary holds great potential. Convenient surveillance requires easy-to-use, cost-effective, ultra-portable but highly reliable, in-vitro-diagnostic devices that are ready for use wherever they are needed. Currently, there are not yet such devices available on the market, but there are a couple more promising technologies developed at readiness-level 5: the Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) lateral-flow-strip tests and the Single-Molecule-with-a-large-Transistor (SiMoT) bioelectronic palmar devices. They both hold key features delineated by the World-Health-Organization for POCT systems and an occurrence of false-positive and false-negative errors <1–5% resulting in diagnostic-selectivity and sensitivity >95–99%, while limit-of-detections are of few markers. CRISPR-strip is a molecular assay that, can detect down to few copies of DNA/RNA markers in blood while SiMoT immunometric and molecular test can detect down to a single oligonucleotide, protein marker, or pathogens in 0.1mL of blood, saliva, and olive-sap. These technologies can prospectively enable the systematic and reliable surveillance of asymptomatic ones prior to worsening/proliferation of illnesses allowing for timely diagnosis and swift prognosis. This could establish a proactive healthcare ecosystem that results in effective treatments for all living organisms generating diffuse and well-being at efficient costs.
