Digital microfluidics is introduced as a novel platform with unique advantages for performing single-molecule detection. We demonstrate how superparamagnetic beads, used for capturing single protein molecules, can be printed with unprecedentedly high loading efficiency and single bead resolution on an electrowetting-on-dielectric-based digital microfluidic chip by micropatterning the Teflon-AF surface of the device. By transporting droplets containing suspended superparamagnetic beads over a hydrophilic-in-hydrophobic micropatterned Teflon-AF surface, single beads are trapped inside the hydrophilic microwells due to their selective wettability and tailored dimensions. Digital microfluidics presents the following advantages for printing and sealing magnetic beads for single-molecule detection: (i) droplets containing suspended beads can be transported back and forth over the array of hydrophilic microwells to obtain high loading efficiencies of microwells with single beads, (ii) the use of hydrophilic-in-hydrophobic patterns permits the use of a magnet to speed up the bead transfer process to the wells, while the receding droplet meniscus removes excess beads off the chip surface and thereby shortens the bead patterning time, and (iii) reagents can be transported over the printed beads multiple times, while capillary forces and a magnet hold the printed beads in place. High loading efficiencies (98% with a CV of 0.9%) of single beads in microwells were obtained by transporting droplets of suspended beads over the array 10 times in less than 1 min, which is much higher than previously reported methods (40-60%), while the total surface area needed for performing single-molecule detection can be decreased. The performance of the device was demonstrated by fluorescent detection of the presence of the biotinylated enzyme β-galactosidase on streptavidin-coated beads with a linear dynamic range of 4 orders of magnitude ranging from 10 aM to 90 fM.

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Major advancements in molecular biology and clinical diagnostics cannot be brought about strictly through the use of genomics based methods. Improved methods for protein detection and proteomic screening are an absolute necessity to complement to wealth of information offered by novel, high-throughput sequencing technologies. Only then will it be possible to advance insights into clinical processes and to characterize the importance of specific protein biomarkers for disease detection or the realization of “personalized medicine”. Currently however, large-scale proteomic information is still not as easily obtained as its genomic counterpart, mainly because traditional antibody-based technologies struggle to meet the stringent sensitivity and throughput requirements that are required whereas mass-spectrometry based methods might be burdened by significant costs involved. However, recent years have seen the development of new biodetection strategies linking nucleic acids with existing antibody technology or replacing antibodies with oligonucleotide recognition elements altogether. These advancements have unlocked many new strategies to lower detection limits and dramatically increase throughput of protein detection assays. In this review, an overview of these new strategies will be given.

A new, ultra‐sensitive genetic screening tool based on a fiber‐optic (FO) surface plasmon biosensor and gold‐labeled gene probes is presented. The assay allows for the detection and location of single‐point mutations (SNP) at significantly reduced cost and high speed. The assay is evaluated for SNP detection of 3 different DNA targets, including a GC‐rich cancer gene fragment and a long bacterial sequence. The data proves that the accuracy of the FO‐SNP test is as good or, in some applications, better than classical high‐resolution melting (HRM).

Detection of single nucleotide polymorphisms (SNPs) is a crucial challenge in the development of a novel generation of diagnostic tools. Accurate detection of SNPs can prove elusive, as the impact of a single variable nucleotide on the properties of a target sequence is limited, even if this sequence consists of only a few nucleotides. New, accurate and facile strategies for the detection of point mutations are therefore absolutely necessary for the increased adoption of point-of-care molecular diagnostics. Currently, PCR and sequencing are mostly applied for diagnosing SNPs. However these methods have serious drawbacks as routine diagnostic tools because of their labour intensity and cost. Several new, more suitable methods can be applied to enable sensitive detection of mutations based on specially designed hybridization probes, mutation recognizing enzymes and thermal denaturation. Here, an overview is presented of the most recent advances in the field of fast and sensitive SNP detection assays with strong potential for integration in point-of-care tests.