Single-Cell Detection of Erwinia amylovora Using Bio-Functionalized SIS Sensor.

Herein, we developed a bio-functionalized solution-immersed silicon (SIS) sensor at the single-cell level to identify Erwinia amylovora (E. amylovora), a highly infectious bacterial pathogen responsible for fire blight, which is notorious for its rapid spread and destructive impact on apple and pear orchards. This method allows for ultra-sensitive measurements without pre-amplification or labeling compared to conventional methods. To detect a single cell of E. amylovora, we used Lipopolysaccharide Transporter E (LptE), which is involved in the assembly of lipopolysaccharide (LPS) at the surface of the outer membrane of E. amylovora, as a capture agent. We confirmed that LptE interacts with E. amylovora via LPS through in-house ELISA analysis, then used it to construct the sensor chip by immobilizing the capture molecule on the sensor surface modified with 3'-Aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The LptE-based SIS sensor exhibited the sensitive and specific detection of the target bacterial cell in real time. The dose-response curve shows a linearity (R2 > 0.992) with wide dynamic ranges from 1 to 107 cells/mL for the target bacterial pathogen. The sensor showed the value change (dΨ) of approximately 0.008° for growing overlayer thickness induced from a single-cell E. amylovora, while no change in the control bacterial cell (Bacillus subtilis) was observed, or negligible change, if any. Furthermore, the bacterial sensor demonstrated a potential for the continuous detection of E. amylovora through simple surface regeneration, enabling its reusability. Taken together, our system has the potential to be applied in fields where early symptoms are not observed and where single-cell or ultra-sensitive detection is required, such as plant bacterial pathogen detection, foodborne pathogen monitoring and analysis, and pathogenic microbial diagnosis.

A Membrane Filter-Assisted Mammalian Cell-Based Biosensor Enabling 3D Culture and Pathogen Detection.

We have developed a membrane filter-assisted cell-based biosensing platform by using a polyester membrane as a three-dimensional (3D) cell culture scaffold in which cells can be grown by physical attachment. The membrane was simply treated with ethanol to increase surficial hydrophobicity, inducing the stable settlement of cells via gravity. The 3D membrane scaffold was able to provide a relatively longer cell incubation time (up to 16 days) as compared to a common two-dimensional (2D) cell culture environment. For a practical application, we fabricated a cylindrical cartridge to support the scaffold membranes stacked inside the cartridge, enabling not only the maintenance of a certain volume of culture media but also the simple exchange of media in a flow-through manner. The cartridge-type cell-based analytical system was exemplified for pathogen detection by measuring the quantities of toll-like receptor 1 (TLR1) induced by applying a lysate of P. aeruginosa and live E. coli, respectively, providing a fast, convenient colorimetric TLR1 immunoassay. The color images of membranes were digitized to obtain the response signals. We expect the method to further be applied as an alternative tool to animal testing in various research areas such as cosmetic toxicity and drug efficiency.

Normal-incidence type solution immersed silicon (SIS) biosensor for ultra-sensitive, label-free detection of cardiac troponin I.

Early diagnosis of acute myocardial infarction (AMI) significantly reduce the mortality rate and can be achieved via high-sensitive detection of AMI specific cardiac troponin I (cTnI) biomarker. Here, we present normal-incident type solution-immersed silicon (NI-SIS) ellipsometric biosensor, designed for ultra-high sensitive, high-throughput, label-free detection of the target protein. The NI-SIS sensors are equipped with a specially designed prism that maintains the angle of incidence close to the Brewster angle during operation, which significantly reduces SIS noise signals induced by the refractive index fluctuations of the surrounding medium, improves the signal-to-noise ratio, in-results lowers the detection limit. We applied NI-SIS biosensor for ultra-sensitive detection of cTnI biomarkers in human serum. The optimized sensor chip fabrication and detection operation procedures are proposed. The wide linear concentration ranges of fg/mL to ng/mL is achieved with the detection limit of 22.0 fg/mL of cTnI. The analytical correlation was assessed by linear regression analysis with the results of the Pathfast reference system. These impressive biosensing capabilities of NI-SIS technology have huge potentials for accurate detection of target species in different application areas, such as diagnosis, drug discovery, and food contaminations.

PAS1-modified optical SIS sensor for highly sensitive and specific detection of toluene.

We report on a novel solution immersed silicon (SIS) sensor modified with bio-receptor to detect toluene. To perform this approach, bio-receptor PAS1 which specifically interacts with toluene was chosen as a capture agent for SIS ellipsometric sensing. We constructed wild PAS1 and mutant PAS1 (F46A and F79Y) which are toluene binding-defective. Especially, we utilized an easily accessible capturing approach based on silica binding peptide (SBP) for direct immobilization of PAS1 on the SiO2 surfaces. After the immobilization of SBP-tagged PAS1 to the sensing layers, PAS1-based SIS sensor was evaluated for its ability to recognize toluene. As a result, a significant up-shift in Psi (Ψ) was clearly observed with a low limit of detection (LOD) of 0.1 µM, when treated with toluene on wild PAS1-surface, but not on mutant PAS1-sensing layers, indicating the selective interactions between PAS1 and toluene molecule. The PAS1-SIS sensor showed no changes in Psi (Ψ), if any, negligible, when exposed to benzene, phenol, xylene and 4-nitrophenol as negative controls, thereby demonstrating the specificity of interaction between PAS1 and toluene. Taken together, our results strongly indicate that PAS1-modified ellipsometry sensor can provide a high fidelity system for the accurate and selective detection of toluene.

Current Technologies of Electrochemical Immunosensors: Perspective on Signal Amplification.

An electrochemical immunosensor employs antibodies as capture and detection means to produce electrical charges for the quantitative analysis of target molecules. This sensor type can be utilized as a miniaturized device for the detection of point-of-care testing (POCT). Achieving high-performance analysis regarding sensitivity has been one of the key issues with developing this type of biosensor system. Many modern nanotechnology efforts allowed for the development of innovative electrochemical biosensors with high sensitivity by employing various nanomaterials that facilitate the electron transfer and carrying capacity of signal tracers in combination with surface modification and bioconjugation techniques. In this review, we introduce novel nanomaterials (e.g., carbon nanotube, graphene, indium tin oxide, nanowire and metallic nanoparticles) in order to construct a high-performance electrode. Also, we describe how to increase the number of signal tracers by employing nanomaterials as carriers and making the polymeric enzyme complex associated with redox cycling for signal amplification. The pros and cons of each method are considered throughout this review. We expect that these reviewed strategies for signal enhancement will be applied to the next versions of lateral-flow paper chromatography and microfluidic immunosensor, which are considered the most practical POCT biosensor platforms.

Label-free detection of hepatitis B virus using solution immersed silicon sensors.

Highly sensitive solution immersed silicon (SIS) biosensors were developed for detection of hepatitis B virus (HBV) infection in the early stage. The ultrasensitivity for overlayer thickness at the nonreflecting condition for the p-polarized wave is the basis of SIS sensing technology. The change in thickness due to biomolecular interactions and change in refractive index of the surrounding buffer medium were assessed simultaneously using two separate ellipsometric parameters (Ψ and Δ), respectively, from a single sensing spot. A direct antigen-antibody affinity assay was used to detect and quantify hepatitis B surface antigen (HBsAg), which is the early stage biomarker for HBV infection. The detection limit of 10 pg/ml was achieved for HBsAg in the human blood serum, which is comparable with the results of enzyme-linked immunosorbent assay and other hybrid assays. The SIS sensor's response time was less than 10 min. The SIS sensors exhibit excellent stability and high signal-to-noise ratio, and are cost-effective, which makes them a suitable candidate for point-of-care applications.

Semi-continuous, real-time monitoring of protein biomarker using a recyclable surface plasmon resonance sensor.

Although label-free immunosensors based on, for example, surface plasmon resonance (SPR) provide advantages of real-time monitoring of the analyte concentration, its application to routine clinical analysis in a semi-continuous manner is problematic because of the high cost of the sensor chip. The sensor chip is in most cases regenerated by employing an acidic pH. However, this causes gradual deterioration of the activity of the capture antibody immobilized on the sensor surface. To use sensor chips repeatedly, we investigated a novel surface modification method that enables regeneration of the sensor surface under mild conditions. We introduced a monoclonal antibody (anti-CBP Ab) that detects the conformational change in calcium binding protein (CBP) upon Ca2+ binding (>1mM). To construct a regenerable SPR-based immunosensor, anti-CBP Ab was first immobilized on the sensor surface, and CBP conjugated to the capture antibody (specific for creatine kinase-MB isoform (CK-MB); CBP-CAb) then bound in the presence of Ca2+. A serum sample was mixed with the detection antibody to CK-MB, which generated an SPR signal proportional to the analyte concentration. After each analysis, the sensor surface was regenerated using medium (pH 7) without Ca2+, and then adding fresh CBP-CAb in the presence of Ca2+ for the subsequent analysis. Analysis of multiple samples using the same sensor was reproducible at a rate >98.7%. The dose-response curve was linear for 1.75-500.75ng/mL CK-MB, with an acceptable coefficient of variation of <8.8%. The performance of the immunosensor showed a strong correlation with that of the Pathfast reference system (R2>96%), and exhibited analytical stability for 1 month. To our knowledge, this is the first report of a renewal of a sensor surface with fresh antibody after each analysis, providing high consistency in the assay during a long-term use (e.g., a month at least).

Ultrasensitive, label-free detection of cardiac biomarkers with optical SIS sensor.

Acute myocardial infarction (MI) is the leading cause of high mortality and morbidity rate worldwide, early and accurate diagnosis can increase the chances of survival. In this work, we report a simple, ultrasensitive, label-free, and high-throughput solution immersed silicon (SIS) immunosensor based on non-reflection condition (NRC) for p-polarized wave for early diagnosis of MI. SIS sensor chips are just a thin dielectric polymer layer on the silicon surface, which can be functionalized for specific application. At NRC, SIS sensors are extremely sensitive to the growing thickness of a bio-layer on the sensor surface while independent of refractive index change of the surrounding medium. Therefore, SIS signal is free from thermal noise, unlike surface plasmon resonance based sensor. Also, there is no need of reference signal which facilitates fast and accurate interaction measurement. Here, SIS technology is applied to tackle two issues in MI diagnosis: high sensitivity with the direct assay and the ability to measure in human serum. Myoglobin, creatine kinase-MB, and cardiac troponin I (cTnI) proteins were used as the MI biomarkers. We were able to measure over a broad concentration range with the detection limit of 5 and 10pg/ml for cTnI in PBS and blood serum, respectively. The response time is about 5min. This novel technique is a suitable candidate for cost effective point-of-care application.

Universal evaluation of combined standard uncertainty for rotating-element spectroscopic ellipsometers.

We present for the first time a universal expression for the combined standard uncertainty for all types of rotating-element spectroscopic ellipsometers (RE-SEs). Specifically, we introduce general model functions as universal analytic expressions for the combined standard uncertainties of the ellipsometric sample parameters. The model functions are expressed as functions of influencing quantities that are not known exactly. The detailed expressions for the model functions are provided for the common RE-SEs. Our approach can be used for instrumentation, standardization, simulation, metrology, optimization of measurement conditions, and performance comparison between RE-SEs.

Universal evaluations and expressions of measuring uncertainty for rotating-element spectroscopic ellipsometers.

We obtain the universal evaluations and expressions of measuring uncertainty for all types of rotating-element spectroscopic ellipsometers. We introduce a general data-reduction process to represent the universal analytic functions of the combined standard uncertainties of the ellipsometric sample parameters. To solve the incompleteness of the analytic expressions, we formulate the estimated covariance for the Fourier coefficient means extracted from the radiant flux waveform using a new Fourier analysis. Our approach can be used for optimization of measurement conditions, instrumentation, simulation, standardization, laboratory accreditation, and metrology.

Stokes vector measurement based on snapshot polarization-sensitive spectral interferometry.

This paper describes a Stokes vector measurement method based on a snapshot polarization-sensitive spectral interferometry. We measure perpendicular linearly polarized complex wave information of an anisotropic object in the spectral domain from which an accurate Stokes vector can be extracted. The proposed Stokes vector measurement method is robust to the object plane 3-D pose variation and external noise, and it provides a reliable snapshot solution in numerous spectral polarization-related applications.

Continuous immunosensing of myoglobin in human serum as potential companion diagnostics technique.

To attain early diagnosis of acute myocardial infarction (AMI) with enhanced accuracy, continuous immunosensing has been investigated to measure myoglobin concentration in real-time. To this end, a capture antibody showing rapid reaction kinetics was immobilized on the surface of a surface plasmon resonance sensor. Three problems associated with the continuous sensing of myoglobin in human serum needed to be overcome: non-specific binding of the analyte, aggregation of serum components, and drift of the sensor baseline. Non-specific binding was controlled by pretreating the sample with a detergent mixture consisting of sodium dodecyl sulfate and P20, and adjusting the micelle size and net charge. Aggregation was managed by inactivating certain serum constituents through chelation of heavy metals. Baseline drift perceived in the sensorgram was able to be corrected by compensating for the slope calculated by a linear regression. Under the optimal conditions, the continuous sensor reproducibly traced the varying doses of myoglobin over about 8h with periodic one-point calibration every 3h. The dose-response curve of the sensor was linear with acceptable variations (CVs<4.91% in average) between the detection limit (31.0 ng/mL) and about 2000 ng/mL in the arithmetic scale (R(2)>0.98), covering the clinical concentration range. The immunosensor performance correlated with the Pathfast reference system (R(2)>0.98) and analytical consistency could be maintained for longer than a month if appropriately calibrated. Such immunosensing could be used as a companion diagnostic means along with real-time electrocardiographic measurement, significantly enhancing the sensitivity of AMI diagnosis and thereby enabling treatment at an early stage.

Optimizing the precision of a multichannel three-polarizer spectroscopic ellipsometer.

We developed a multichannel three-polarizer spectroscopic ellipsometer based on a data acquisition algorithm for achieving optimized precision. This algorithm measures unnormalized Fourier coefficients accurately and precisely. Offset angles for optical elements were obtained as wavelength-independent values using regression calibration. Derived subsets of data reduction functions were used to calculate sample parameters. Correlation coefficients of Fourier coefficients were used to calculate errors in the sample parameters. Mean standard deviations of the sample parameters for each data reduction method were compared to identify the best method. This approach could be used to identify suitable precision optimization methods for other rotating-element ellipsometers.

Snapshot phase sensitive scatterometry based on double-channel spectral carrier frequency concept.

Spectroscopic ellipsometry is one of the most important measurement schemes used in the optical nano-metrology for not only thin film measurement but also nano pattern 3D structure measurement. In this paper, we propose a novel snap shot phase sensitive normal incidence spectroscopic ellipsometic scheme based on a double-channel spectral carrier frequency concept. The proposed method can provide both Ψ(λ) and Δ(λ) only by using two spectra acquired simultaneously through the double spectroscopic channels. We show that the proposed scheme works well experimentally by measuring a binary grating with nano size 3D structure. We claim that the proposed scheme can provide a snapshot spectroscopic ellipsometric parameter measurement capability with moderate accuracy.

Study of cell-matrix adhesion dynamics using surface plasmon resonance imaging ellipsometry.

The interaction of cells with extracellular matrix, termed cell-matrix adhesions, importantly governs multiple cellular phenomena. Knowledge of the functional dynamics of cell-matrix adhesion could provide critical clues for understanding biological phenomena. We developed surface plasmon resonance imaging ellipsometry (SPRIE) to provide high contrast images of the cell-matrix interface in unlabeled living cells. To improve the contrast and sensitivity, the null-type imaging ellipsometry technique was integrated with an attenuated total reflection coupler. We verified that the imaged area of SPRIE was indeed a cell-matrix adhesion area by confocal microscopy imaging. Using SPRIE, we demonstrated that three different cell types exhibit distinct features of adhesion. SPRIE was applied to diverse biological systems, including during cell division, cell migration, and cell-cell communication. We imaged the cell-matrix anchorage of mitotic cells, providing the first label-free imaging of this interaction to our knowledge. We found that cell-cell communication can alter cell-matrix adhesion, possibly providing direct experimental evidence for cell-cell communication-mediated changes in cell adhesion. We also investigated shear-stress-induced adhesion dynamics in real time. Based on these data, we expect that SPRIE will be a useful methodology for studying the role of cell-matrix adhesion in important biological phenomena.

Fourier analysis for rotating-element ellipsometers.

We introduce a Fourier analysis of the waveform of periodic light-irradiance variation to capture Fourier coefficients for multichannel rotating-element ellipsometers. In this analysis, the Fourier coefficients for a sample are obtained using a discrete Fourier transform on the exposures. The analysis gives a generic function that encompasses the discrete Fourier transform or the Hadamard transform, depending on the specific conditions. Unlike the Hadamard transform, a well-known data acquisition method that is used only for conventional multichannel rotating-element ellipsometers with line arrays with specific readout-mode timing, this Fourier analysis is applicable to various line arrays with either nonoverlap or overlap readout-mode timing. To assess the effects of the novel Fourier analysis, the Fourier coefficients for a sample were measured with a custom-built rotating-polarizer ellipsometer, using this Fourier analysis with various numbers of scans, integration times, and rotational speeds of the polarizer.

Angle-resolved annular data acquisition method for microellipsometry.

An ellipsometric data acquisition method is introduced to measure the optical properties of sample. It is based on a microellipsometer hardware layout integrated a high numerical aperture objective lens, which is aligned in the normal direction of sample surface. This technique enables to achieve ellipsometric data at multiple incident angle with a sub-mum probe beam size, moreover real-time measurement is possible due to no moving parts. The experimental results of different SiO(2) thin film are demonstrated, also calibration technique is described.