Biodiagnostic Devices Research Articles

Functionalized nanoporous gold as a new biosensor platform for ultra-low quantitative detection of human serum albumin

The development of a new generation of ultra-sensitive sensors for analytical and bio-diagnostic devices requires a strong signal in front of very small quantity of analyte, often present in complex and interfering matrix. Taking advantage of peculiar plasmonic properties of nanoporous gold (NPG), a promising sensor for selective detection of Human Serum Albumin (HSA), a proof-of-concept bioanalyte, was prepared and tested in a conventional micro-Raman spectrometer using Surface Enhanced Raman Spectroscopy (SERS). NPG was synthesized by chemical de-alloying of an amorphous precursor, Au20Cu48Ag7Pd5Si20, starting from melt spun ribbons. A fully de-alloyed ribbon with ligaments of about 60 nm was obtained after 4 h of de-alloying at 70 °C and 10 M HNO3 + 0.5 M HF. This material is self-standing, mechanically resistant, and suitable for wide range of applications. At this stage, NPG is SERS-active toward several molecules, including pyridine, bi-pyridine, and rhodamine at very low concentration. In order to obtain the selective binding properties required to detect molecules in bio-diagnostic applications, immuno-functionalization of NPG was carried out by using an anti-HSA antibody (Ab-anti-HSA) covalently grafted on gold ligaments via the 4-aminothiophenol (4ATP) Raman probe, yielding the SERS-active nanohybrid NPG-4ATP-Ab. SERS signal recorded at increasing HSA concentrations was used for the acquisition of SERS maps and a chemometric regression model allowed to calibrate the sensor. In the best experimental conditions, we were able to get quantitative analysis of HSA at ultra-low concentrations (0.1 ng/l) with SERS.The methodology proposed in this paper has to be considered a major step toward a calibrated device for ultra-sensitive detection of biomolecules by SERS detection.

Silk-Encapsulated Plasmonic Biochips with Enhanced Thermal Stability.

Because of their high sensitivity, cost-efficiency, and great potential as point-of-care biodiagnostic devices, plasmonic biosensors based on localized surface plasmon resonance have gained immense attention. However, most plasmonic biosensors and conventional bioassays rely on natural antibodies, which are susceptible to elevated temperatures and nonaqueous media. Hence, an expensive and cumbersome "cold chain" system is necessary to preserve the labile antibodies by maintaining optimal cold temperatures during transport, storage, and handling. Herein, we introduce a facile approach to preserve the antibody activity on a biosensor surface even at elevated temperatures. We show that silk fibroin film could be used as a protective layer to preserve the activity of a model antibody (Rabbit IgG) and cardiac troponin antibody at both room temperature and 40 °C over several days. Furthermore, a simple aqueous rinsing process restores the biofunctionality of the biosensor. This energy-efficient and environmentally friendly method represents a novel approach to eliminate the cold chain and temperature-controlled packing of diagnostic reagents and materials, thereby extending the capability of antibody-based biosensors to different resource-limited circumstances such as developing countries, an ambulance, an intensive care unit emergency room, and battlefield.

Magnetic nanobeads present during enzymatic amplification and labeling for a simplified DNA detection protocol based on AC susceptometry

Magnetic biosensors are promising candidates for low-cost point-of-care biodiagnostic devices. For optimal efficiency it is crucial to minimize the time and complexity of the assay protocol includi ...

Paper-based plasmon-enhanced protein sensing by controlled nucleation of silver nanoparticles on cellulose

Cheap, disposable bio-diagnostic devices are becoming increasingly prevalent in the field of biosensing. Earlier we had reported the ability of cellulosic surface to control the nucleation of plasmonic silver nanoparticles and in this report we utilize this nucleation controlling property to demonstrate a new plasmonic sensing mechanism based on paper substrates to quantitatively detect proteins. On contrary to conventional paper based diagnostic devices which use the cellulosic part of paper as a support structure, the proposed method takes advantage of cellulose as nucleation controller during silver nanoparticle formation. Reduction of silver ions interacting competitively with nucleation controlling cellulosic surface and reduction suppressing amino acids of protein (via complexation) resulted in silver nanoparticles whose size–shape dependent plasmonic property quantitatively reflected the concentration of protein on paper, characterized using UV–Vis and surface-enhanced Raman spectroscopies. As a proof-of-concept, bovine serum albumin (BSA) was tested as the target analyte. UV–Vis spectroscopy based BSA quantification was sensitive in the concentration range 10–60 mg ml−1 while that for surface enhanced Raman spectroscopy extended well below 10 mg ml−1, thus demonstrating the potential of this simple method to quantitatively detect a wide range of proteins relevant to the field of biodiagnostics.

Biomarker Binding on an Antibody-Functionalized Biosensor Surface: The Influence of Surface Properties, Electric Field, and Coating Density

Antibodies have been used as bioreceptors in biodiagnostic devices for decades, the performances of which are affected by various factors, such as orientation, density, and local environment. While there are extensive works on design and fabrication of various biosensors, little is known about the molecular level interactions between antibodies coated on sensor surfaces and biomarkers suspended in medium. In this work, a coarse-grained model for biomarkers binding on an antibody-functionalized biosensor surface is constructed to study the effects of surface properties and external parameters on antibody orientation and biomarkers binding time. The surface interaction type is found to significantly influence the antibody orientation and biomarker binding time. A proper electric field range is discovered to not only well-orient antibodies but also steer biomarkers toward the surface, consequently reducing the binding time of biomarkers by 2 orders of magnitude. Moreover, a suitable surface coating density of antibodies has been proposed to help antibody orientation as well as biomarker binding. These findings can be used for rational design of biosensors with higher efficiency and more sensitive detections.

Polymethyl methacrylate-co-methacrylic acid coatings with controllable concentration of surface carboxyl groups: A novel approach in fabrication of polymeric platforms for potential bio-diagnostic devices

The generally accepted strategy in development of bio-diagnostic devices is to immobilize proteins on polymeric surfaces as a part of detection process for diseases and viruses through antibody/antigen coupling. In that perspective, polymer surface properties such as concentration of functional groups must be closely controlled in order to preserve the protein activity. In order to improve the surface characteristics of transparent polymethacrylate plastics that are used for diagnostic devices, we have developed an effective fabrication procedure of polymethylmetacrylate-co-metacrylic acid (PMMA-co-MAA) coatings with controlled number of surface carboxyl groups. The polymers were processed effectively with the spin-coating technique and the detailed control over surface properties is here by demonstrated through the variation of a single synthesis reaction parameter. The chemical structure of synthesized and processed co-polymers has been investigated with nuclear magnetic resonance spectroscopy (NMR) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-ToF-MS). The surface morphology of polymer coatings have been analyzed with atomic force microscopy (AFM) and scanning electron microscopy (SEM). We demonstrate that the surface morphology and the concentration of surface –COOH groups (determined with UV–vis surface titration) on the processed PMMA-co-MAA coatings can be precisely controlled by variation of initial molar ratio of reactants in the free-radical polymerization reaction. The wettability of developed polymer surfaces also varies with macromolecular structure.

Droplet Microfluidic Chip Based Nucleic Acid Amplification and Real-Time Detection of Influenza Viruses

Miniaturized bio-diagnostic devices have the potential to allow for rapid pathogen screening in clinical patient samples, as a low cost and portable alternative to conventional bench-top equipment. Miniaturization of key bio-diagnostic techniques, such as: nucleic acid detection and quantification, polymerase chain reaction (PCR), DNA fingerprinting, enzyme linked immunosorbent assay (ELISA), results in substantial reduction of reaction volumes (expensive samples/reagents) and shorter reaction times. Droplet microfluidics (DMF) is one of several miniaturized bio-sample handling techniques available for manipulating clinical samples and reagents in microliter (10−6 L) to picoliter (10−12 L) volume regime. Electro-actuation of sample and reagent in the form of droplets in the aforementioned volume regime, using dielectrophoresis (DEP) and/or Electrowetting (EW) are achieved by means of patterned, insulated metal electrodes on one or more substrates. In this work, we have utilized electro-actuation based DMF technology, integrated with suitably tailored resistive micro-heaters and temperature sensors, to achieve chip based real-time, quantitative PCR (qRT-PCR). This qRT-PCR micro-device was utilized to detect and quantify the presence of influenza A and C virus nucleic acids, using in-vitro synthesized viral RNA segments. The experimental analysis of the DMF micro-device confirms its capabilities in qRT-PCR based detection and quantification of pathogen samples, with accuracy levels comparable to established commercial bench-top equipment (PCR efficiency ∼95%). The limit of detection (LOD) of the chip based qRT-PCR technique was estimated to be ∼5 copies of template RNA per PCR reaction.

A magnetic nanobead‐based bioassay provides sensitive detection of single‐ and biplex bacterial DNA using a portable AC susceptometer

Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin–biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM–NDA further towards implementation in point-of-care and outpatient settings.

Glycodendrimers: versatile tools for nanotechnology

Combining nanotechnology with glycobiology has triggered an exponential growth of research activities in the design of novel functional bionanomaterials (glyconanotechnology). More specifically, recent synthetic advances towards the tailored and versatile design of glycosylated nanoparticles namely glyconanoparticles, considered as synthetic mimetics of natural glycoconjugates, paved the way toward diverse biomedical applications. The accessibility of a wide variety of these structured nanosystems, in terms of shapes, sizes, and organized around stable nanoparticles have readily contributed to their development and applications in nanomedicine. In this context, glycosylated gold-nanoparticles (GNPs), glycosylated quantum dots (QDs), fullerenes, single-wall natotubes (SWNTs), and self-assembled glycononanoparticles using amphiphilic glycopolymers or glycodendrimers have received considerable attention to afford powerful imaging, therapeutic, and biodiagnostic devices. This review will provide an overview of the most recent syntheses and applications of glycodendrimers in glycoscience that have permitted to deepen our understanding of multivalent carbohydrate-protein interactions. Together with synthetic breast cancer vaccines, inhibitors of bacterial adhesions to host tissues including sensitive detection devices, these novel bionanomaterials are finding extensive relevance.