Early cancer diagnosis requires ultrasensitive detection of tumor markers in blood. To this end, we develop a novel microcantilever immunosensor using nanobodies (Nbs) as receptors. As the smallest antibody (Ab) entity comprising an intact antigen-binding site, Nbs achieve dense receptor layers and short distances between antigen-binding regions and sensor surfaces, which significantly elevate the generation and transmission of surface stress. Owing to the inherent thiol group at the C-terminus, Nbs are covalently immobilized on microcantilever surfaces in directed orientation via one-step reaction, which further enhances the stress generation. For microcantilever-based nanomechanical sensor, these advantages dramatically increase the sensor sensitivity. Thus, Nb-functionalized microcantilevers can detect picomolar concentrations of tumor markers with three orders of magnitude higher sensitivity, when compared with conventional Ab-functionalized microcantilevers. This proof-of-concept study demonstrates an ultrasensitive, label-free, rapid, and low-cost method for tumor marker detection. Moreover, interestingly, we find Nb inactivation on sensor interfaces when using macromolecule blocking reagents. The adsorption-induced inactivation is presumably caused by the change of interfacial properties, due to binding site occlusion upon complex coimmobilization formations. Our findings are generalized to any coimmobilization methodology for Nbs and, thus, for the construction of high-performance immuno-surfaces. Electronic Supplementary Material Supplementary material (experimental section, HER2 detection using anti-HER2-mAb-functionalized microcantilevers) is available in the online version of this article at 10.1007/s12274-021-3588-4.

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The Advisory Committee on Cancer for the World Health Organization suggests that "a third of cancers are curable if diagnosed early". [3] Traditional methods of cancer diagnosis are based primarily on histo-pathological examination and imaging of the tumor tissue, which are the current gold standards. [4] However, these methods tend to detect specific lesions only when they are formed in the middle and later stages of cancer development, and are not suitable for screening early cancers in a large population. In the early stage of cancer development, before the formation of specific lesions, cancer cells in the body begin to release a few cancer markers related to cancer formation and metastasis. [5] At this time, if a highly sensitive detection method can be available to detect the very low concentration of markers in body fluids, it will bring great potential for the early detection and diagnosis of cancer. In addition, traditional methods are invasive, and inaccurate biopsy positioning can cause sampling errors. X-ray screening and histo-pathological evaluation rely heavily on the individual's expertise, which causes discrepancies between individual and laboratory-reported results. [6] All these limitations indicate the need for highly sensitive and minimally invasive techniques for early diagnosis and treatment of cancers. In comparison, liquid biopsies can offer early discovery and a non-invasive approach.In recent years, cancer markers in body fluids, such as circulating tumor-derived proteins (carcinoembryonic antigen, prostate-specific antigen), circulating tumor cells, and circulating tumor DNA, [7] have been the most common biomarkers for cancer diagnosis and assessment of treatment efficacy. However, these biomarkers have several shortcomings in early-stage cancer diagnosis. Circulating tumor-derived protein markers are compromised by high false positive rates, which can lead to overdiagnosis and, in some cases, to unnecessary anticancer treatment. [8] The fraction of circulating tumor DNA in total circulating free DNA is usually very low and often < 0.01%; therefore, this can complicate early detection. [9] Circulating tumor cells are tumor cells that have detached from the primary tumor, with extremely low quantities. Exosomes are a class of nanoscale vesicles secreted by cells, which contain abundant information closely related to parental cells. The ultrasensitive detection of cancer-derived exosomes is highly significant for early noninvasive diagnosis of cancer.Here, an ultrasensitive nanomechanical sensor is reported, which uses a magnetic-driven microcantilever array to selectively detect oncogenic exosomes. A magnetic force, which can produce a far greater deflection of microcantilever than that produced by the intermolecular interaction force even with very low concentrations of target substances, is introduced. This method reduced the detection limit to less than 10 exosomes mL −1 . Direct detection of exosomes in the serum of patients with breast cancer and in healthy people showed a signifi...