Advanced Analysis of Biosensor Data for SARS-CoV-2 RBD and ACE2 Interactions.

The traditional approach for analyzing interaction data from biosensors instruments is based on the simplified assumption that also larger biomolecules interactions are homogeneous. It was recently reported that the human receptor angiotensin-converting enzyme 2 (ACE2) plays a key role for capturing SARS-CoV-2 into the human target body, and binding studies were performed using biosensors techniques based on surface plasmon resonance and bio-layer interferometry. The published affinity constants for the interactions, derived using the traditional approach, described a single interaction between ACE2 and the SARS-CoV-2 receptor binding domain (RBD). We reanalyzed these data sets using our advanced four-step approach based on an adaptive interaction distribution algorithm (AIDA) that accounts for the great complexity of larger biomolecules and gives a two-dimensional distribution of association and dissociation rate constants. Our results showed that in both cases the standard assumption about a single interaction was erroneous, and in one of the cases, the value of the affinity constant KD differed more than 300% between the reported value and our calculation. This information can prove very useful in providing mechanistic information and insights about the mechanism of interactions between ACE2 and SARS-CoV-2 RBD or similar systems.

Precision improvement in dark-field microscopy imaging by using gold nanoparticles as an internal reference: a combined theoretical and experimental study.

Low accuracy is a big obstacle in the dark-field microscopy imaging (iDFM) technique in practical applications. In order to reduce the deviations and fluctuations in the observed or snapped scattered light in the iDFM technique caused by unavoidable measurement errors, bare gold nanoparticles (AuNPs) were introduced as an internal reference (IR). The feasibility of using AuNPs as the IR in iDFM in theory was verified. The function of the IR in improving the precision of the acquired data through post data analysis was identified by three kinds of experiments: monitoring the oxidation process of silver nanoparticles (AgNPs) at room temperature, quantifying the level of glucose with AgNPs used as probes and quantifying the change in the light intensity of AuNPs after the plasmon resonance energy transfer (PRET) between AuNPs and tetramethylrhodamine (TAMRA).

Quantitative blood group typing using surface plasmon resonance.

The accurate and reliable typing of blood groups is essential prior to blood transfusion. While current blood typing methods are well established, results are subjective and heavily reliant on analysis by trained personnel. Techniques for quantifying blood group antibody-antigen interactions are also very limited. Many biosensing systems rely on surface plasmon resonance (SPR) detection to quantify biomolecular interactions. While SPR has been widely used for characterizing antibody-antigen interactions, measuring antibody interactions with whole cells is significantly less common. Previous studies utilized SPR for blood group antigen detection, however, showed poor regeneration causing loss of functionality after a single use. In this study, a fully regenerable, multi-functional platform for quantitative blood group typing via SPR detection is achieved by immobilizing anti-human IgG antibody to the sensor surface, which binds to the Fc region of human IgG antibodies. The surface becomes an interchangeable platform capable of quantifying the blood group interactions between red blood cells (RBCs) and IgG antibodies. As with indirect antiglobulin tests (IAT), which use IgG antibodies for detection, IgG antibodies are initially incubated with RBCs. This facilitates binding to the immobilized monolayer and allows for quantitative blood group detection. Using the D-antigen as an example, a clear distinction between positive (>500 RU) and negative (<100 RU) RBCs is achieved using anti-D IgG. Complete regeneration of the anti-human IgG surface is also successful, showing negligible degradation of the surface after more than 100 regenerations. This novel approach is validated with human-sourced whole blood samples to demonstrate an interesting alternative for quantitative blood grouping using SPR analysis.

The simultaneous detection of free and total prostate antigen in serum samples with high sensitivity and specificity by using the dual-channel surface plasmon resonance.

Free/total prostate antigen (f/t-PSA) ratio in serum as a promising parameter has been used to improve the differentiation of benign and malignant prostate disease. In order to obtain the accurate and reliable f/t-PSA ratio, the simultaneous detection of f-PSA and t-PSA with high sensitivity and specificity is required. In this work, the dual-channel surface plasmon resonance (SPR) has been employed to meet the requirement. In one channel, t-PSA was directly measured with a linear range from 1.0 to 20.0 ng/mL. In another channel, due to the low concentration of f-PSA in serum, the asynchronous competitive inhibition immunoassay with f-PSA@Au nanoparticles (AuNPs) was developed. As expected, the detection sensitivity of f-PSA was greatly enhanced, and a linear correlation with wider linear range from 0.010 to 0.40 ng/mL was also achieved. On the other hand, a simple method was explored for significantly reducing the non-specific adsorption of co-existing proteins. On basis of this, the f/t-PSA ratios in serum samples from prostate cancer (PCa) or benign prostatic hyperplasia (BPH) patients were measured. And it was found that there was significant difference between the distributions of f/t-PSA ratio in BPH patients (16.44±1.77%) and those in PCa patients (24.53±4.97%). This present work provides an effective method for distinguishing PCa from BPH, which lays a potential foundation for the early diagnosis of PCa.

Ultra-broadband wide-angle unidirectional plasmonic coupler based on joint effects of plasmonic critical angles and subwavelength metallic gratings.

A unidirectional plasmonic coupler with ultra-broadband and wide-angle coupling efficiency is proposed and demonstrated. Employing the plasmonic critical angle (PCA) phenomenon in conjunction with the effect of subwavelength metallic gratings, a wide incident angular full-width-half-maximum (AFWHM >28°) coupling efficiency larger than 50% and extinction ratio better than 15 dB over an ultrabroad wavelength range 725-985 nm (bandwidth >260 nm) are achieved. The operation principle and performance of the structure are clarified and theoretically illustrated.

A reusable localized surface plasmon resonance biosensor for quantitative detection of serum squamous cell carcinoma antigen in cervical cancer patients based on silver nanoparticles array.

Squamous cell carcinoma antigen (SCCa), as a tumor biomarker, plays an important role in adjuvant diagnosis, treatment evaluation, and prognosis prediction for cervical cancer patients. Localized surface plasmon resonance (LSPR) technique based on noble metal nanoparticles bypasses the disadvantages of traditional testing strategies, in terms of free-labeling, short assay time, good sensitivity, and selectivity. Herein, we develop a novel and reusable LSPR biosensor for the detection of SCCa. First, a triangle-shaped silver nanoparticle array was fabricated using the nanosphere lithography method. Next, we investigated and verified the feasibility of amino coupling method using 11-mercaptoundecanoic acid (MUA) to form a functionalized chip surface with monoclonal anti-SCCa antibodies on the silver nanoparticles for distinct detection of SCCa. Different concentrations of SCCa were successfully tested in both buffer and human serum by the ultrasensitive and specific LSPR system, with a linear quantitative detection range of 0.1-1,000 pM under optimal conditions. With appropriate regeneration solution, for example 50 mM glycine-HCl (pH 2.0), the LSPR biosensor featured effective fabrication reproducibility, which reduced both production cost and testing time. Our study represents the first application of the LSPR biosensor in cervical cancer, and demonstrates that the rapid, simple, and reusable nanochip can serve as a potential alternative for clinical serological diagnosis of SCCa in cervical cancer patients.

Enhancement of field-analyte interaction at metallic nanogap arrays for sensitive localized surface plasmon resonance detection.

We investigated the near-field enhancement of a localized surface plasmon resonance (LSPR) structure based on gold nanograting pairs with a nanosized gap. The results calculated by finite-difference time-domain and rigorous coupled-wave analysis methods presented that the nanogap enclosed by two neighboring nanogratings produced significant confinement and enhancement of electromagnetic fields and allowed a sensitive detection in sensing of surface binding events. Gold gratings with a narrow gap distance less than 10 nm showed enhanced refractive index sensitivity due to the intensified optical field at the nanogap, outperforming the LSPR structure with noninteracting nanogratings. Also, we analyzed the effectiveness of using an overlap integral (OI) between analyte and local plasmon field to estimate the detection sensitivity. We found a strong correlation of field-analyte OI with far-field sensor sensitivity.

MIRG Survey 2011: snapshot of rapidly evolving label-free technologies used for characterizing molecular interactions.

The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions.

Sensitive detection of unlabeled oligonucleotides using a paired surface plasma waves biosensor.

Detection of unlabeled oligonucleotides using surface plasmon resonance (SPR) is difficult because of the oligonucleotides' relatively lower molecular weight compared with proteins. In this paper, we describe a method for detecting unlabeled oligonucleotides at low concentration using a paired surface plasma waves biosensor (PSPWB). The biosensor uses a sensor chip with an immobilized probe to detect a target oligonucleotide via sequence-specific hybridization. PSPWB measures the demodulated amplitude of the heterodyne signal in real time. In the meantime, the ratio of the amplitudes between the detected output signal and reference can reduce the excess noise from the laser intensity fluctuation. Also, the common-path propagation of p and s waves cancels the common phase noise induced by temperature variation. Thus, a high signal-to-noise ratio (SNR) of the heterodyne signal is detected. The sequence specificity of oligonucleotide hybridization ensures that the platform is precisely discriminating between target and non-target oligonucleotides. Under optimized experimental conditions, the detected heterodyne signal increases linearly with the logarithm of the concentration of target oligonucleotide over the range 0.5-500 pM. The detection limit is 0.5 pM in this experiment. In addition, the non-target oligonucleotide at concentrations of 10 pM and 10nM generated signals only slightly higher than background, indicating the high selectivity and specificity of this method. Different length of perfectly matched oligonucleotide targets at 10-mer, 15-mer and 20-mer were identified at the concentration of 150 pM.

Oligopeptides functionalized surface plasmon resonance biosensors for detecting thiacloprid and imidacloprid.

By using phage display library, we identified two highly specific oligopeptide sequences RKRIRRMMPRPS and RNRHTHLRTRPR for binding neonicotinoids such as thiacloprid and imidacloprid. The former shows high affinity for thiacloprid whereas the latter shows high affinity for imidacloprid. Surprisingly, cross binding is minimal despite the similarity of the two molecules. To develop a neonicotinoid biosensor, these two oligopeptides are synthesized and immobilized on the surface of a surface plasmon resonance (SPR) chip with a bare-gold surface. This oligopeptide functionalized SPR biosensor can rapidly detect thiacloprid and imidacloprid in buffer solutions in a real-time manner. The limit of detection (LOD) for thiacloprid and imidacloprid is 1.2 µM and 0.9 µM, respectively.

A S-parameters-based detection method for a multilayer SPR biosensor.

In this paper, S-parameters investigation of a variable incidence angle multilayer SPR biosensor is presented. Both magnitude and phase of the S-parameters are taken into account in the investigation. The work presented in this paper is the first attempt to apply S-parameters analysis to a multilayer SPR biosensor. The goal is to improve sensitivity through involving S-parameters including their phase values. In addition, further investigation is carried out to understand the relationship between the S-parameters and thickness of biomolecular layer and also the design parameters including the number of graphene layers.

Improved sensitivity of wavelength-modulated surface plasmon resonance biosensor using gold nanorods.

In this report, gold nanorods (GNRs) were used to enhance the sensitivity of the wavelength-modulated surface plasmon resonance (SPR) biosensor. The GNRs were designed and fabricated through seed-medicated growth and surface activation by a layer of a weak polyelectrolyte, poly(acrylic acid) for the attaching antibody. Rabbit anti-goat IgG was immobilized on GNRs, and sandwich assays were carried out to detect goat IgG using a wavelength-modulated SPR biosensor. The detection sensitivity of the nanorod-conjugated antibody is 25-100 times more sensitive than the SPR biosensor without GNRs. Drastic sensitivity enhancement, owing to the electromagnetic interaction between the nanotag and the sensing film, was maximized using the longitudinal plasmonic resonance of the GNRs. GNRs could significantly enhance the sensitivity of the SPR biosensor, and the maximum enhancement effect can be achieved when the longitudinal SPR peak wavelength of GNRs functionally matches the surface plasmon wavelength.

Emerging role of surface plasmon resonance in fragment-based drug discovery.

Surface plasmon resonance (SPR) offers a method of biophysical fragment screening that is fast, efficient, cost effective and accurate. SPR is increasingly being adopted as a secondary assay to validate fragment hits. Recently, technical advances have resulted in the emergence of SPR as a primary screening methodology for fragment-based drug discovery. Moreover, SPR biosensor assays can be developed for a wide range of proteins, including membrane proteins, such as G-protein-coupled receptors. In this review, we discuss the advantages and limitations of SPR fragment screening including experimental consideration of reducing false positive and false negative rates to a minimum. We discuss how ligand efficiency can be used both as a method to eliminate false positives and to understand which fragments in a library may be a source of false negatives.

Poly(HEMA) brushes emerging as a new platform for direct detection of food pathogen in milk samples.

Surface plasmon resonance (SPR) biosensors capable of in real time detection of Cronobacter at concentrations down to 106 cells mL⁻¹ in samples of consumer fresh-whole fat milk, powder whole-fat milk preparation, and powder infant formulation were developed for the first time. Antibodies against Cronobacter were covalently attached onto polymer brushes of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) grafted from the SPR chip surface. The lowest detection limit, 104 cells mL⁻¹, was achieved in phosphate buffered saline (pH 7.4) with sensors prepared by covalent immobilization of the same antibodies onto a self assembled monolayer (SAM) of hexa(ethylene glycol) undecanethiol (EG6). However, when the EG6 based sensors were challenged with milk samples the non-specific response due to the deposition of non-targeted compounds from the milk samples was much higher than the specific response to Cronobacter hampering the detection in milk. Similar interfering fouling was observed on antifouling polymer brushes of hydroxy-capped oligoethylene glycol methacrylate and even a 10 times higher fouling was observed on the widely used SAM of mixed hydroxy- and carboxy-terminated alkanethiols. Only poly(HEMA) brushes totally suppressed the fouling from milk samples. The robust well-controlled surface initiated atom transfer radical polymerization of HEMA allowed the preparation of highly dense brushes with a minimal thickness so that the capture of antigens by the antibodies immobilized on the brush layer could take place close to the gold SPR surface to provide a stronger optical response while the fouling was still suppressed. A minimum thickness of 19 nm of poly(HEMA) brush layer was necessary to suppress completely non-specific sensor response to fouling from milk.

One-chip biosensor for simultaneous disease marker/calibration substance measurement in human urine by electrochemical surface plasmon resonance method.

We have developed a miniaturized electrochemical surface plasmon resonance biosensor for measuring two biomolecules that have very different molecular sizes, one is transferrin (MW=75 kDa) as a disease marker protein, the other is creatinine (MW=113) as a calibration marker for the accurate measurement of human urinary samples. The sensor has a PDMS based microchannel that is 2 mm wide and 20 µm deep. Two gold films were integrated in the microchannel; one was modified with anti-transferrin antibody for immuno-reaction, and the other was modified with osmium-poly-vinylpyridine wired horseradish peroxidase (Os-gel-HRP). We further immobilized a tri-enzyme layer of creatininase, creatinase and sarcosine oxidase in order to measure creatinine by converting it to hydrogen peroxide in the upstream channel. We measured the transferrin concentration from the refractive index change involved in an immuno-complex formation, and we were simultaneously able to measure creatinine by employing the refractive index change in the Os-gel-HRP caused by oxidation with the hydrogen peroxide produced from creatinine by the tri-enzyme. The effects of ascorbic acid and uric acid in urine samples were sufficiently eliminated by adding ascorbate oxidase and uricase to the urine samples during sampling. We were able to measure two analyte concentrations within 15 min by one simple injection of 50 µL of diluted human urine into our sensor. The detectable transferrin and creatinine ranges were 20 ng/mL to 10 µg/mL, and 10 µM to 10 mM, respectively, which are sufficient levels for clinical tests. Finally, we compared the results obtained using our sensor with those obtained with a conventional immunoassay and the Jaffe method. We obtained a similar trend that can reduce the fluctuation in the urinary transferrin concentration from three different samples by calibrating the creatinine concentration.

Improving immunosensor performances using an acoustic mixer on droplet microarray.

A major drawback of protein microarrays is the lack of control of ligand immobilization at the surface of the chip which limits their performances and thus their impacts in in vitro diagnosis. To improve antibody (Ab) grafting during the spotting process on commercialized gold SPRi chips, we propose to produce a chaotic flow in every spotted droplet, by using an acoustic field, in order to disrupt the steady state of the reaction of Ab grafting. Our results show that acoustic mixing during Ab binding at the biochips surface increases their biorecognition performances of a mean factor of 2.7 in comparison with Ab layer grafted in a passive mode. Moreover, it increases statistically the homogeneity of the response over all the surface of the chips.

Fiber-optic particle plasmon resonance sensor for detection of interleukin-1ß in synovial fluids.

A facile and label-free biosensing method has been developed for determining an osteoarthritis concerned cytokine, interleukin-1ß (IL-1ß), in synovial fluids. The biosensing technique, fiber-optic particle plasmon resonance (FOPPR), is based on gold nanoparticles-modified optical fiber where the gold nanoparticle surface has been modified by a mixed self-assembled monolayer for further conjugation of anti-IL-1ß antibody and minimization of nonspecific adsorption. Upon binding of IL-1ß to anti-IL-1ß on the gold nanoparticle surface, the absorbance of the gold nanoparticle layer on the optical fiber changes and the signal change is enhanced through multiple total internal reflections along the optical fiber. Results show that the detection of IL-1ß in synovial fluid by this sensor agrees quantitatively with the clinically accepted enzyme-linked immunosorbent assay (ELISA) method but a much shorter analysis time is required (<10 min). The sensor response versus log concentration of IL-1ß was linear (r=0.9947) over the concentration range of 0.050-10 ng/mL and a limit of detection (LOD) of 21 pg/mL (1.2 pM) was achieved. Such a LOD for IL-1ß (17 kDa) represents a major advancement in the field of real-time monitoring of low molecular weight proteins in complex biological fluids.

Detection of swine-origin influenza A (H1N1) viruses using a localized surface plasmon coupled fluorescence fiber-optic biosensor.

Swine-origin influenza A (H1N1) virus (S-OIV) was identified as a new reassortant strain of influenza A virus in April 2009 and led to an influenza pandemic. Accurate and timely diagnoses are crucial for the control of influenza disease. We developed a localized surface plasmon coupled fluorescence fiber-optic biosensor (LSPCF-FOB) which combines a sandwich immunoassay with the LSP technique using antibodies against the hemagglutinin (HA) proteins of S-OIVs. The detection limit of the LSPCF-FOB for recombinant S-OIV H1 protein detection was estimated at 13.9 pg/mL, which is 10(3)-fold better than that of conventional capture ELISA when using the same capture antibodies. For clinical S-OIV isolates measurement, meanwhile, the detection limit of the LSPCF-FOB platform was calculated to be 8.25 × 10(4)copies/mL, compared with 2.06 × 10(6)copies/mL using conventional capture ELISA. Furthermore, in comparison with the influenza A/B rapid test, the detection limit of the LSPCF-FOB for S-OIV was almost 50-fold in PBS solution and 25-fold lower in mimic solution, which used nasal mucosa from healthy donors as the diluent. The findings of this study therefore indicate that the high detection sensitivity and specificity of the LSPCF-FOB make it a potentially effective diagnostic tool for clinical S-OIV infection and this technique has the potential to be applied to the development of other clinical microbe detection platforms.

Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma.

Surface plasmon resonance (SPR) biosensor for high-throughput screening of protein biomarkers in diluted blood plasma is reported. The biosensor combines a high-resolution SPR imaging sensor and a high-density protein array with low-fouling background. The SPR imaging sensor utilizes polarization contrast and advanced referencing and provides a total of 120 sensing areas (each 200 µm×150 µm). Antibodies are immobilized on the sensing areas via hybridization of antibody-oligonucleotide conjugates to thiolated complementary oligonucleotides microspotted on the sensor surface (DNA-directed immobilization). A low-fouling background is achieved by covalent immobilization of bovine serum albumin to carboxyl-terminated thiols filling the areas among the thiolated oligonucleotides and outside the sensing areas. The biosensor was evaluated for detection of protein biomarkers relevant to cancer diagnostics--human chorionic gonadotropin (hCG) and activated leukocyte cell adhesion molecule (ALCAM) both in buffer and in 10% blood plasma. Limits of detection as low as 45 ng/mL (ALCAM) and 100 ng/mL (hCG) were achieved in blood plasma samples.

Differential spectral phase interferometry for wide dynamic range surface plasmon resonance biosensing.

We introduce a novel wide dynamic range phase-sensitive surface plasmon resonance (SPR) biosensor based on differential spectral interferometry. Superseding conventional spectroscopic approach where only the SPR dip is monitored, our system acquires the spectral phase information of the entire electromagnetic field that undergoes SPR transformation. Since the SPR-induced phase change is highly wavelength specific with fixed incident angle, ultra-high sensitivity achievable through phase-sensitive detection, as reported herein, is maintained continuously across the spectral domain in response to refractive index changes. Our system has demonstrated a detection limit of 2.2×10(-7) in terms of refractive index unit (RIU) using standard single-layer gold surface. In terms of biosensing performance, the estimated detection sensitivity obtained from bovine serum albumin (BSA) antibody-antigen binding experiments is 0.5 ng ml(-1).