Surface plasmon resonance (SPR) biosensors are an advanced optical biosensing technology that has been widely used in molecular biology for the investigation of biomolecular interactions and in bioanalytics for the detection of biological species. This work aims to review progress in the development of SPR biosensors for medical diagnostics, focusing mainly on advances in optical platforms and assays enabling analysis of complex biological matrices. Applications of SPR biosensors for the detection of medically relevant analytes, such as nucleic acids, proteins, exosomes, viruses, bacteria, and circulating tumor cells, are also reviewed. The detection performance of current SPR biosensors is discussed, and routes for improving performance and expanding applications of SPR biosensors in medical diagnostics are outlined.
- Keywords
- Biomarker, Medical diagnostics, Optical biosensor, Plasmonic affinity biosensor, Surface plasmon resonance biosensor,
- MeSH
- Bacteria isolation & purification MeSH
- Biosensing Techniques * instrumentation methods MeSH
- Equipment Design MeSH
- Exosomes MeSH
- Humans MeSH
- Neoplasms diagnosis MeSH
- Nucleic Acids isolation & purification analysis MeSH
- Surface Plasmon Resonance * instrumentation methods MeSH
- Proteins isolation & purification analysis MeSH
- Viruses isolation & purification MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Nucleic Acids MeSH
- Proteins MeSH
We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.
- Keywords
- biosensor, immunoassays, rolling circle amplification, single molecule, surface plasmon resonance, surface plasmon-enhanced fluorescence,
- MeSH
- Biosensing Techniques * methods MeSH
- DNA, Single-Stranded MeSH
- Limit of Detection MeSH
- Surface Plasmon Resonance methods MeSH
- Nucleic Acid Amplification Techniques * methods MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- DNA, Single-Stranded MeSH
Functional gold nanoparticles (AuNPs) are commonly used to enhance the response of optical affinity biosensors. In this work, we investigated the effect of preparation conditions on functional properties of AuNPs functionalized with antibody (Ab-AuNPs), specifically AuNPs with antibody against carcinoembryonic antigen (CEA) covalently attached via carboxy-terminated oligo-ethylene thiolate linker layer. The following parameters of preparation of Ab-AuNP have been found to have a significant effect on Ab-AuNP performance in affinity biosensors: the time of reaction of activated AuNPs with antibody, concentrations of antibody and amino-coupling reagents, and composition of immobilization buffer (molarity and salt content). In contrast, pH of immobilization buffer has been demonstrated to have only a minor influence. Our experiments showed that the Ab-AuNPs prepared under optimum conditions offered a binding efficiency of Ab-AuNPs to CEA as high as 63%, which is more than 4 times better than the best efficiencies reported for similar functional AuNPs so far. We employed these Ab-AuNPs with a surface plasmon resonance (SPR) biosensor for the detection of CEA and showed that the Ab-AuNPs enhanced the sensor response to CEA by a factor of 1000. We also demonstrated that the Ab-AuNPs allow the biosensor to detect CEA at concentrations as low as 12 and 40 pg/mL in buffer and 50% blood plasma, respectively.
- Keywords
- Antibody, Cancer marker carcinoembryonic antigen, Functionalization, Gold nanoparticles, Optical affinity biosensor, Surface plasmon resonance,
- MeSH
- Antibodies, Immobilized chemistry MeSH
- Carcinoembryonic Antigen analysis blood MeSH
- Hydrogen-Ion Concentration MeSH
- Metal Nanoparticles chemistry MeSH
- Humans MeSH
- Limit of Detection MeSH
- Surface Plasmon Resonance methods MeSH
- Buffers MeSH
- Gold chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Names of Substances
- Antibodies, Immobilized MeSH
- Carcinoembryonic Antigen MeSH
- Buffers MeSH
- Gold MeSH
Label-free affinity biosensors offer a promising platform for the development of a new generation of medical diagnostic technologies. Nevertheless, when such sensors are used in complex biological media, adsorption of non-targeted medium components prevents the specific detection of the analyte. In this work, we introduce for the first time a biosensor assay based on surface plasmon resonance (SPR) capable of diagnosing different stages of Epstein-Barr virus (EBV) infections in clinical serum samples. This was achieved by simultaneous detection of the antibodies against three different antigens present in the virus. To prevent the interference of the fouling from serum during the measurement, the SPR chips were coated by an antifouling layer of a polymer brush of poly[oligo(ethylene glycol) methacrylate] grown by surface-initiated atom transfer radical polymerization. The bioreceptors were then attached via hybridization of complementary oligonucleotides. This allowed the sensor surface to be regenerated after measurement by disrupting the complementary pairs above the oligonucleotides' melting temperature and attaching new bioreceptors. In this way, the same sensing surface could be used repeatedly. The procedure used in this work will serve as a prototype strategy for the development of label-free affinity biosensors for diagnostics in blood serum or plasma samples. This is the first example of detection of marker of a disease in clinical serum samples by an optical affinity biosensor.
- Keywords
- Antifouling, Epstein–Barr virus infection, Polymer brushes, Real time diagnostics, Surface plasmon resonance biosensor,
- MeSH
- Equipment Failure Analysis MeSH
- Biosensing Techniques instrumentation MeSH
- Equipment Design MeSH
- Immunoassay instrumentation MeSH
- Epstein-Barr Virus Infections blood diagnosis immunology MeSH
- Humans MeSH
- Surface Plasmon Resonance instrumentation MeSH
- Antibodies, Viral immunology MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Antibodies, Viral MeSH
The study of optical affinity biosensors based on plasmonic nanostructures has received significant attention in recent years. The sensing surfaces of these biosensors have complex architectures, often composed of localized regions of high sensitivity (electromagnetic hot spots) dispersed along a dielectric substrate having little to no sensitivity. Under conditions such that the sensitive regions are selectively functionalized and the remaining regions passivated, the rate of analyte capture (and thus the sensing performance) will have a strong dependence on the nanoplasmonic architecture. Outside of a few recent studies, there has been little discussion on how changes to a nanoplasmonic architecture will affect the rate of analyte transport. We recently proposed an analytical model to predict transport to such complex architectures; however, those results were based on numerical simulation and to date, have only been partially verified. In this study we measure the characteristics of analyte transport across a wide range of plasmonic structures, varying both in the composition of their base plasmonic element (microwires, nanodisks, and nanorods) and the packing density of such elements. We functionalized each structure with nucleic acid-based bioreceptors, where for each structure we used analyte/receptor sequences as to maintain a Damköhler number close to unity. This method allows to extract both kinetic (in the form of association and dissociation constants) and analyte transport parameters (in the form of a mass transfer coefficient) from sensorgrams taken from each substrate. We show that, despite having large differences in optical characteristics, measured rates of analyte transport for all plasmonic structures match very well to predictions using our previously proposed model. These results highlight that, along with optical characteristics, analyte transport plays a large role in the overall sensing performance of a nanoplasmonic biosensor.
Early diagnosis of ongoing malignant disease is crucial to improve survival rate and life quality of the patients and requires sensitive detection of specific biomarkers e.g. prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), etc. In spite of current technological advances, malignant diseases are still identified in rather late stages, which have detrimental effect on the prognosis and treatment of the disease. Here, we present a biosensor able to detect fetuin-A, a potential multibiomarker. The biosensing platform is based on polymer brush combining antifouling monomer units of N-(2-hydroxypropyl)methacrylamide (HPMA) and carboxybetaine methacrylamide (CBMAA), statistically copolymerized by surfaceinitiated atom transfer radical polymerization. The copolymer poly(HPMA-co-CBMAA) exhibits excellent non-fouling properties in the most relevant biological media (i.e. blood plasma) as well as antithrombogenic surface properties by preventing the adhesion of blood components (i.e. leukocytes; platelets; and erythrocytes). Moreover, the polymer brush can be easily functionalized with biorecognition elements maintaining high resistance to blood fouling and the binding capacity can be regulated by tuning the ratio between CBMAA and HPMA units. The superior antifouling properties of the copolymer even after biofunctionalization were exploited to fabricate a new plasmonic biosensor for the analysis of fetuin-A in real clinical blood plasma samples. The assay used in this work can be explored as labelfree affinity biosensor for diagnostics of different biomarkers in real clinical plasma samples and to shift the early biomarker detection toward novel biosensor technologies allowing point of care analysis.
- MeSH
- Biomarkers blood MeSH
- Biosensing Techniques methods MeSH
- alpha-2-HS-Glycoprotein analysis metabolism MeSH
- Humans MeSH
- Surface Plasmon Resonance methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Biomarkers MeSH
- alpha-2-HS-Glycoprotein MeSH
The surface plasmon resonance (SPR) biosensor system with dispersionless microfluidics for the direct and label-free detection of a soluble vascular endothelial growth factor receptor (sVEGFR-1) is described. The detection approach takes advantage of an affinity interaction between sVEGFR-1 and its ligand, vascular endothelial growth factor (VEGF-A), which is covalently immobilized on the surface of the SPR sensor. The ability of the immobilized VEGF-A to specifically bind the sVEGFR-1 receptor is demonstrated in a buffer. The detection of sVEGFR-1 in 2% human blood plasma is carried out by using the sequential injection approach. The detection limit of 25 ng/mL is achieved. In addition, we demonstrate that the functional surface of the sensor can be regenerated for repeated use.
- MeSH
- Biomarkers blood MeSH
- Biosensing Techniques methods MeSH
- Humans MeSH
- Myelodysplastic Syndromes blood diagnosis metabolism MeSH
- Surface Plasmon Resonance methods MeSH
- Vascular Endothelial Growth Factor Receptor-1 blood metabolism MeSH
- Vascular Endothelial Growth Factor A blood metabolism MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Biomarkers MeSH
- Vascular Endothelial Growth Factor Receptor-1 MeSH
- Vascular Endothelial Growth Factor A MeSH
Affinity-based biosensing systems have become an important analytical tool for the detection and study of numerous biomolecules. The merging of these sensing technologies with microfluidic flow cells allows for faster detection times, increased sensitivities, and lower required sample volumes. In order to obtain a higher degree of performance from the sensor, it is important to know the effects of the flow cell geometry on the sensor sensitivity. In these sensors, the sensor sensitivity is related to the overall diffusive flux of analyte to the sensing surface; therefore increases in the analyte flux will be manifested as an increase in sensitivity, resulting in a lower limit of detection (LOD). Here we present a study pertaining to the effects of the flow cell height H on the analyte flux J, where for a common biosensor design we predict that the analyte flux will scale as J ≈ H(-2/3). We verify this scaling behavior via both numerical simulations as well as an experimental surface plasmon resonance (SPR) biosensor. We show the reduction of the flow cell height can have drastic effects on the sensor performance, where the LOD of our experimental system concerning the detection of ssDNA decreases by a factor of 4 when H is reduced from 47 μm to 7 μm. We utilize these results to discuss the applicability of this scaling behavior with respect to a generalized affinity-based biosensor.
- MeSH
- DNA, Single-Stranded analysis genetics MeSH
- Limit of Detection MeSH
- Microfluidic Analytical Techniques instrumentation MeSH
- Surface Plasmon Resonance instrumentation MeSH
- Base Sequence MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Single-Stranded MeSH
In this study we examine the experimental use of the staggered herringbone mixer (SHM) for the signal enhancement of a microfluidic surface plasmon resonance imaging (SPRi) affinity-based biosensor. We define the signal enhancement (Emix) as the ratio of the time-dependent slope of the sensor response of a SHM-based microfluidic channel and that of an unmixed channel; Emix is directly proportional to changes in the sensor sensitivity and inversely proportional to changes in the sensor limit of detection (LOD). Measurements were carried out for three SHM designs under a wide range of volumetric flow rates for two analytes: high diffusivity ssDNA and low diffusivity Escherichia coli bacteria. The experimental data collected in this study was found to exhibit a good match to that predicted by the numerical methods discussed in part I of this study. We found that Emix is dependent on the SHM groove geometry, the Péclet number Pe, and the overall microchannel length L; these dependencies are discussed in detail. For realistic experimental conditions, the enhancement that the SHM can provide is in the range of 1 < Emix < 5 (0% < improvement < 400%).
- MeSH
- Models, Biological MeSH
- Biosensing Techniques instrumentation MeSH
- Escherichia coli isolation & purification MeSH
- DNA, Single-Stranded analysis MeSH
- Microfluidic Analytical Techniques instrumentation MeSH
- Surface Plasmon Resonance MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Single-Stranded MeSH
We report on the use of new biofunctionalized gold nanoparticles (bio-AuNPs) that enable a surface plasmon resonance (SPR) biosensor to detect low levels of carcinoembryonic antigen (CEA) in human blood plasma. Bio-AuNPs consist of gold nanoparticles functionalized both with (1) streptavidin, to provide high affinity for the biotinylated secondary antibody used in the second step of the CEA sandwich assay, and with (2) bovine serum albumin, to minimize the nonspecific interaction of the bio-AuNPs with complex samples (blood plasma). We demonstrate that this approach makes it possible for the SPR biosensor to detect CEA in blood plasma at concentrations as low as 0.1 ng/mL, well below normal physiological levels (approximately nanograms per milliliter). Moreover, the limit of detection achieved using this approach is better by a factor of more than 1,000 than limits of detection reported so far for CEA in blood plasma using SPR biosensors.
- MeSH
- Biotinylation MeSH
- Immobilized Proteins chemistry MeSH
- Carcinoembryonic Antigen blood immunology MeSH
- Humans MeSH
- Limit of Detection MeSH
- Nanoparticles chemistry MeSH
- Surface Plasmon Resonance methods MeSH
- Antibodies chemistry immunology MeSH
- Serum Albumin, Bovine chemistry MeSH
- Cattle MeSH
- Streptavidin chemistry MeSH
- Gold chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Immobilized Proteins MeSH
- Carcinoembryonic Antigen MeSH
- Antibodies MeSH
- Serum Albumin, Bovine MeSH
- Streptavidin MeSH
- Gold MeSH
