Real-Time FO-SPR Monitoring of Solid-Phase DNAzyme Cleavage Activity for Cutting-Edge Biosensing

DNA nanotechnology has a great potential in biosensor design including nanostructuring of the biosensor surface through DNA origami, target recognition by means of aptamers, and DNA-based signal amplification strategies. In this paper, we use DNA nanotechnology to describe for the first time the concept of real-time solid-phase monitoring of DNAzyme cleavage activity for the detection of specific single-stranded DNA (ssDNA) with a fiber optic surface plasmon resonance (FO-SPR) biosensor. Hereto, we first developed a robust ligation strategy for the functionalization of the FO-SPR biosensing surface with ssDNA-tethered gold nanoparticles, serving as the substrate for the DNAzyme. Next, we established a relation between the SPR signal change, due to the cleavage activity of the 10–23 DNAzyme, and the concentration of the DNAzyme, showing faster cleavage kinetics for higher DNAzyme concentrations. Finally, we implemented this generic concept for biosensing of ssDNA target in solution. Hereto, we designed a DNAzyme–inhibitor complex, consisting of an internal loop structure complementary to the ssDNA target, that releases active DNAzyme molecules in a controlled way as a function of the target concentration. We demonstrated reproducible target detection with a theoretical limit of detection of 1.4 nM, proving that the presented ligation strategy is key to a universal DNAzyme-based FO-SPR biosensing concept with promising applications in the medical and agrofood sector

Improved Method for Counting DNA Molecules on Biofunctionalized Nanoparticles

In order to accurately determine low numbers (1−100) of immobilized ssDNA molecules at a single, silica 250 nm nanoparticle surface, we hereby propose an integrated approach combining classic single molecule confocal microscopy (SMCM), that is, stepwise photobleaching of labeled ssDNA, with modified total internal reflection fluorescence microscopy (mTIRF). We postulate that SMCM alone is unable to exactly account for all labeled ssDNA because of inherent laser polarization effects; that is, perpendicularly oriented molecules to the sample surface are not (or are only slightly) susceptible to laser excitation and thus are invisible in a classic photobleaching experiment. The SMCM method accounts for at best two-thirds (68%) of the present ssDNA molecules. The principle of the mTIRF technique, which relies on the creation of highly inclined illumination combined with part of the laser remaining in normal Köhler illumination, enables accurate counting of SMCM invisble molecules. The combined approach proposed here circumvents the polarization issue and allows a complete single molecule counting on individual nanoparticles, fully in line with bulk measurements, as will be demonstrated

Media 1: Flexible tool for simulating the bulk optical properties of polydisperse spherical particles in an absorbing host: experimental validation

Originally published in Optics Express on 25 August 2014 (oe-22-17-20223

Immunoassay for Detection of Infliximab in Whole Blood Using a Fiber-Optic Surface Plasmon Resonance Biosensor

Monitoring the concentration of a therapeutic drug antibody, infliximab (IFX), is recommended for enhancing its efficacy in patients with inflammatory bowel disease (IBD). However, IFX concentrations are currently determined in patients’ serum/plasma, which requires sample preparation from blood, hence hampering the turnaround time. In this paper, we present a short immunoassay (10 min) using a fiber-optic surface plasmon resonance (FO-SPR) biosensor for detection of IFX spiked in 100-fold diluted serum, plasma, and whole blood. The calculated limits of detection (LOD) based on calibration curves were 1.42, 1.00, and 1.34 ng/mL, respectively, which coincides with expected IFX concentrations in diluted samples from IBD patients. A linear correlation was established among different matrixes, indicating that the matrix effect was insignificant. The established point-of-care (POC) FO-SPR bioassay was also used to measure IFX in 100-fold diluted extracts of dried blood spots (DBS), and LOD achieved was below 2 ng/mL. Although DBS might be ideal for POC, this is the first report of using an SPR biosensor for measuring DBS samples. Finally, the POC FO-SPR immunoassay was validated by using matching serum and plasma samples from five IBD patients. A Pearson correlation of 0.968 was obtained between serum and plasma samples. IFX concentrations determined with FO-SPR were compared to a clinically validated enzyme-linked immunosorbent assay (ELISA), resulting in excellent Pearson correlation and intraclass correlation coefficient, both being 0.99 for serum and plasma samples. In conclusion, this paper demonstrates that our FO-SPR biosensor can be used as a true POC diagnostic tool for determining IFX concentrations in a variety of matrixes