Microfluidic quartz-crystal-microbalance (QCM) sensors with specialized immunoassays for extended measurement range and improved reusability

Abstract

Quartz crystal microbalances (QCM) are well known mass sensitive devices. By combining them with specialized antibodies, sensitive biosensors with detection limits down to some nanogram of substance can be obtained. While the antibody coating determines which substances will be detected, the QCM's limit of detection is determined by the required minimal mass change. Therefore, measurements of biological samples containing small target molecules at low concentrations remain challenging. Here, we present the fabrication of disposable but also regenerative QCM devices with microfluidic housing and an approach to improve the detection limit of a QCM by employing an additional, heavier molecule to increase the total added apparent mass, leading to a larger drop of the QCMs resonant frequency per amount of measured substance. As any signal amplification bears the risk to lose its benefit by amplification of unspecific binding too, we further developed an in vitro designed antibody sandwich pair, offering more specific target molecule (CRP) detection and only temporary binding which renders the device reusable within minutes. No additional chemicals or regeneration protocols between measurements are necessary, sole purging with buffer solution is sufficient. To achieve signal amplification, streptavidin was attached via biotin to the second antibody. The resulting frequency drop was up to five times higher compared to the original antibody sandwich. In the absence of CRP, no resonant frequency change rising above signal noise could be observed in presence of the second antibody and streptavidin. The presented concept is broadly expandable. The use of even heavier molecules or nanoparticles for mass amplification should allow higher frequency shifts per amount of target substance and promises to extend the measurement range of QCMs into the sub-nanogramm region.