Continuous biomarker monitoring with single molecule resolution by measuring free particle motion.

There is a need for sensing technologies that can continuously monitor concentration levels of critical biomolecules in applications such as patient care, fundamental biological research, biotechnology and food industry, as well as the environment. However, it is fundamentally difficult to develop measurement technologies that are not only sensitive and specific, but also allow monitoring over a broad concentration range and over long timespans. Here we describe a continuous biomolecular sensing methodology based on the free diffusion of biofunctionalized particles hovering over a sensor surface. The method records digital events due to single-molecule interactions and enables biomarker monitoring at picomolar to micromolar concentrations without consuming any reagents. We demonstrate the affinity-based sensing methodology for DNA-based sandwich and competition assays, and for an antibody-based cortisol assay. Additionally, the sensor can be dried, facilitating storage over weeks while maintaining its sensitivity. We foresee that this will enable the development of continuous monitoring sensors for applications in fundamental research, for studies on organs on a chip, for the monitoring of patients in critical care, and for the monitoring of industrial processes and bioreactors as well as ecological systems.

Reversible Immunosensor for the Continuous Monitoring of Cortisol in Blood Plasma Sampled with Microdialysis.

Cortisol is a steroid hormone involved in a wide range of medical conditions. The level of the hormone fluctuates over time, but with traditional laboratory-based assays, such dynamics cannot be monitored in real time. Here, a reversible cortisol sensor is reported that allows continuous monitoring of cortisol in blood plasma using sampling by microdialysis. The sensor is based on measuring single-molecule binding and unbinding events of tethered particles. The particles are functionalized with antibodies and the substrate with cortisol-analogues, causing binding and unbinding events to occur between particles and substrate. The frequency of binding events is reduced when cortisol is present in the solution as it blocks the binding sites of the antibodies. The sensor responds to cortisol in the high nanomolar to low micromolar range and can monitor cortisol concentrations over multiple hours. Results are shown for cortisol monitoring in filtered and in microdialysis-sampled human blood plasma.

The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling.

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 "CRC genes." These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.

Free-standing hydrogel-particle composite membrane with dynamically controlled permeability.

The preparation and investigation of a free-standing membrane made from a composite of thermoresponsive poly(N-isopropylacrylamide) (pNIPAAm) and polystyrene nanoparticles (PS NP) with temperature-controlled permeability is reported. The method exploits the light-induced crosslinking of the photo-reactive pNIPAAm-based polymer and mechanical reinforcement of the membrane structure by the polystyrene nanoparticles. About micrometer thick layers were either directly attached to a gold surface or prepared as free-standing layers spanning over arrays of microfluidic channels with a width of about hundred microns by using template stripping. Diffusion of liquid medium, low molecular weight molecules, and large molecular weight proteins contained in blood through the composite membrane was observed with combined surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The swelling ratio, permeability, and nonspecific sorption to these composite membranes were investigated by SPR and OWS as a function of molecular weight of analyte, loading of PS NP in the composite film, and temperature. The authors show successful preparation of a defect-free membrane structure that acts as a thermoresponsive filter with nanoscale pores spanning over an area of several square millimeters. This membrane can be reversibly switched to block or allow the diffusion of low mass molecules to the sensor surface by temperature-triggered swelling and collapsing of the hydrogel component. Blocking of diffusion and low unspecific sorption of proteins contained in blood serum is observed. These features make this platform interesting for potential future applications in continuous monitoring biosensors for the analysis of low molecular weight drug analytes or for advanced cell-on-chip microfluidic studies.

Plasmon Field-Enhanced Fluorescence Energy Transfer for Hairpin Aptamer Assay Readout.

Surface plasmon field-enhanced fluorescence energy transfer is employed for sensitive optical readout of a reversible hairpin aptamer assay that is suitable for continuous monitoring of low-molecular-weight chemical analytes. A hairpin aptamer specific to adenosine and adenosine triphosphate with Alexa Fluor 647 fluorophore attached to its 5' end was anchored via 3' end thiol to a gold thin film. Molecular spacers were used to control the distance of the fluorophore from the surface in the aptamer "off" and "on" states. The specific binding of the target analyte changes the aptamer conformation, which alters the distance of the fluorophore from the gold surface and translates to variations in the detected fluorescence intensity. The plasmonically mediated fluorescence signal increases the measured signal-to-noise ratio and allows for real-time observation of the analyte binding. Theoretical as well as experimental study of the optical signal dependence on fluorophore orientation, design of spacers, and angular distribution of collected light is presented for rational design of the assay. The detected sensor signal increased by a factor as large as 23 upon switching the aptamer from the "off" to "on" state due to the hairpin opening associated with the specific capture of target analyte.

Biosensor based on hydrogel optical waveguide spectroscopy for the detection of 17ß-estradiol.

Rapid detection of hormones at sub-ng/ml concentrations is of tremendous importance for diagnostic purposes, quality control, and environmental monitoring. In this respect, we report a novel label-free biosensor based on hydrogel optical waveguide spectroscopy (HOWS) for the sensitive detection of 17ß-estradiol (E2). This approach was implemented by using a thin hydrogel layer of a carboxylated poly(N-isoproprylacrylamide) (PNIPAAm) terpolymer that was attached to a metallic sensor surface in order to simultaneously serve as a binding matrix and an optical waveguide. Refractive index changes that are accompanied with the specific capture of biomolecules from an aqueous sample on the sensor surface were probed by resonantly excited hydrogel optical waveguide modes. To optically excite and interrogate these waves, an optical setup based on Kretschmann configuration of attenuated total reflection (ATR) method that is compatible with surface plasmon resonance (SPR) was used. We demonstrate that HOWS offers a higher binding capacity, good anti-fouling properties, improved figure of merit, and E2 detection limit of 50 pg/ml which is seven times better than that obtained by a regular surface plasmon resonance (SPR) biosensor.

The current status of cancer biomarker research using tumour-associated antigens for minimal invasive and early cancer diagnostics.

Tumour-associated antigens (TAA) can be detected prior to clinical diagnosis and thus would be ideal biomarkers for early detection of cancer using only a few microliters of a patient's serum. In this article we provide a summary of TAA screening and serum-profiling conducted for breast, prostate, lung and colon cancers. Different methodological approaches, including SEREX, SERPA, and phage display for TAA identification and TAA panels are summarised, and a revision of array based techniques is provided. The most promising studies performed on these cancers (performed with 80-400 serum samples, including controls) obtained sensitivities in a range of 44-95% and specificities of 80-100%. From the various studies reviewed, only one performed cross validation (AUC=0.71) in a prostate cancer study. Thus, albeit receiver operation characteristics are very promising, cross validation of most studies is still missing. Additionally, the concerted action of research groups for standardization of serum-TAA testing and cross validation is required. Along with today's technological options, the chances of establishing TAA biomarkers are now higher than ever before. This may also be true for confirmation and validation of already existing data, which is a prerequisite for implementation of TAA biomarkers into clinical diagnostics. This article is part of a Special Issue entitled: Integrated omics.