4-(Triethoxysilyl)butanoic acid as a self-assembled monolayer for surface modification of titanium dioxide.

In this study, the carboxy silane 4-(triethoxysilyl)butanoic acid (TESBA) was used to modify titanium dioxide (TiO2) to create a self-assembled monolayer (SAM) and then directionally immobilize a capture antibody using protein A. We selected the amino silane (3-aminopropyl)triethoxysilane (APTES) to perform a comparative analysis with TESBA, and employed glutaraldehyde (GA) as the control. The modification and detection effects and the limit of detection (LOD) were evaluated by detecting human immunoglobulin G (IgG). The average normalized sensitivity of the dual-grating coupler waveguide biosensor was 49.63 ± 0.27 RIU-1 and the optimum resolution was 1.30 × 10-6 RIU. When the SAM was prepared using TESBA and APTES followed by GA, the LOD was 4.59 × 10-7 g mL-1 and 5.29 × 10-7 g mL-1, respectively. We analyzed the modification and detection effects by the t-test and concluded that the differences in the modification effects using TESBA and APTES followed by GA were significant and the differences in the detection effects using TESBA and APTES followed by GA were insignificant. The use of TESBA as the SAM led to the modification effect being superior to that obtained using APTES followed by GA. The detection effect using TESBA was as outstanding as that using APTES followed by GA. Our findings demonstrate the feasibility and effectiveness of using TESBA as the SAM to carboxylate the surface of TiO2, thereby enabling immobilization of biomolecules for human IgG detection.

Rapid and Highly Sensitive Detection of C-Reaction Protein Using Robust Self-Compensated Guided-Mode Resonance BioSensing System for Point-of-Care Applications.

The rapid and sensitive detection of human C-reactive protein (CRP) in a point-of-care (POC) may be conducive to the early diagnosis of various diseases. Biosensors have emerged as a new technology for rapid and accurate detection of CRP for POC applications. Here, we propose a rapid and highly stable guided-mode resonance (GMR) optofluidic biosensing system based on intensity detection with self-compensation, which substantially reduces the instability caused by environmental factors for a long detection time. In addition, a low-cost LED serving as the light source and a photodetector are used for intensity detection and real-time biosensing, and the system compactness facilitates POC applications. Self-compensation relies on a polarizing beam splitter to separate the transverse-magnetic-polarized light and transverse-electric-polarized light from the light source. The transverse-electric-polarized light is used as a background signal for compensating noise, while the transverse-magnetic-polarized light is used as the light source for the GMR biosensor. After compensation, noise is drastically reduced, and both the stability and performance of the system are enhanced over a long period. Refractive index experiments revealed a resolution improvement by 181% when using the proposed system with compensation. In addition, the system was successfully applied to CRP detection, and an outstanding limit of detection of 1.95 × 10-8 g/mL was achieved, validating the proposed measurement system for biochemical reaction detection. The proposed GMR biosensing sensing system can provide a low-cost, compact, rapid, sensitive, and highly stable solution for a variety of point-of-care applications.

Exterior-electrode electrically driven microconcentrator.

This study uses negative dielectrophoresis and AC electroosmosis as a driving mechanism and presents an electrically driven microconcentrator that concentrates the sample in the region exterior to the electrodes (termed as exterior-electrode electrically driven microconcentrator in this paper). The proposed microconcentrator uses a 3-D face-to-face electrode pair; the top electrode is a relatively large planar electrode, and the bottom electrode is formed with three to six long and thin electrodes connected into an open ring. The sample is brought to the vicinity of the open electrode at the bottom by electroosmotic flow; then, negative dielectrophoresis is used to push the sample away from the electrode and concentrate it in the region surrounded by the open ring electrode. Concentration using an exterior-electrode electrically driven microconcentrator offers promise for convenient use in conjunction with relevant detection systems. The results indicate that for the proposed exterior-electrode electrically driven microconcentrator, the optimal frequency is 100 kHz and the optimal voltage is 13 Vp-p . The corner concentration process at the corners of the bottom open electrodes enables the multi-corner electrodes to exhibit better concentration results than that exhibited by semicircular-shaped electrodes. The concentration performance is most favorable when the shape of the open electrode at the bottom is a five-vertex electrode, enabling a concentration enhancement factor of 55 times for a latex particle solution and 11 times for E. coli. The experimental results also demonstrate that the concentration phenomenon in this study is not induced by non-specific adsorption and can be repeated multiple times.

Enhanced sensitivity in injection-molded guided-mode-resonance sensors via low-index cavity layers.

We present an investigation on the use of low-index cavity layers to enhance the sensitivity of injection-molded guided-mode resonance (GMR) sensors. By adjusting the sputtering parameters, a low-index cavity layer is created at the interface between the waveguide layer and the substrate. Refractive index measurements show that a sensitivity enhancement of up to 220% is achieved with a cavity layer, in comparison to a reference GMR sensor without a cavity layer. Finite-element-method simulations were performed, and the results indicate that the cavities significantly redistribute the resonance mode profile and thus enhances the sensitivity. The present investigation demonstrates a new method for enhancing the sensitivity of injection-molded GMR sensors for high-sensitivity label-free biosensing.

On-line SERS detection of single bacterium using novel SERS nanoprobes and a microfluidic dielectrophoresis device.

The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfluidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica. The SERS nanoprobes are prepared by immobilization of specific antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defined plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identification of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB-DEP-Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL(-1) . In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.

Forced-convective vitrification with liquid cryogens.

Cell cryopreservation by vitrification generally requires using vitrification solutions with high concentrations of cryoprotectants (CPAs), which are toxic and induce osmotic stresses associated with the addition and removal of CPAs. To increase the cooling rate and reduce the CPA concentration required for vitrification, this study proposed an innovative approach, named forced-convective vitrification with liquid cryogens, in which liquid oxygen at a temperature below its boiling point (LOX(bbp)) was used as the cryogen to reduce the generation of insulating bubbles of gaseous oxygen and the sample was subjected to a constant velocity to remove insulation bubbles from the sample. Results show that changing the cryogen from liquid nitrogen at its boiling temperature (LN(abp)) to LOX(bbp), increasing the sample velocity and reducing the test solution volume increased the cooling rate and thereby decreased the CPA concentration required for vitrification. Using the same velocity (1.2 m/s), the cooling rate achieved with LOX(bbp) was 2.3-fold greater than that achieved with LN(abp). With LOX(bbp), the increase in the sample velocity from 0.2 to 1.2 m/s enhanced the cooling rate by 1.9 times. With LOX(bbp), a velocity of 1.2m/s and a test solution volume of 1.73 µl, the CPA concentration required for vitrification decreased to 25%. These results indicate that the new approach described here can reduce the CPA concentration required for vitrification, and thus decreases the toxicity and osmotic stresses associated with adding and removing the CPA.

Using a fiber optic particle plasmon resonance biosensor to determine kinetic constants of antigen-antibody binding reaction.

In this paper, one simple and label-free biosensing method has been developed for determining the binding kinetic constants of antiovalbumin antibody (anti-OVA) and anti-mouse IgG antibody using the fiber optic particle plasmon resonance (FOPPR) biosensor. The FOPPR sensor is based on gold-nanoparticle-modified optical fiber, where the gold nanoparticle surface has been modified by a mixed self-assembled monolayer for conjugation of a molecular probe reporter (ovalbumin or mouse IgG) to dock with the corresponding analyte species such as anti-OVA or anti-mouse IgG. The binding process, occurring when an analyte reacts with a probe molecule immobilized on the optical fiber, can be monitored in real-time. In addition, by assuming a Langmuir-type adsorption isotherm to measure the initial binding rate, the quantitative determination of binding kinetic constants, the association and dissociation rate constants, yields k(a) of (7.21 ± 0.4) × 10(3) M(-1) s(-1) and k(d) of (2.97 ± 0.1) × 10(-3) s(-1) for OVA/anti-OVA and k(a) of (1.45 ± 0.2) × 10(6) M(-1) s(-1) and k(d) of (2.97 ± 0.6) × 10(-2) s(-1) for mouse IgG/anti-mouse IgG. We demonstrate that the FOPPR biosensor can study real-time biomolecular interactions.

Cryopreserved chondrocytes in porous biomaterials with surface elastin and poly-L-lysine for cartilage regeneration.

The ability of cryopreserved chondrocytes to revitalize and propagate is a key biotechnology in cartilage regeneration. This study shows the formation of neocartilage from cryopreserved chondrocytes in scaffolds grafted with elastin and poly-L-lysine. Cryopreserved chondrocytes in elastin- and poly-L-lysine-grafted constructs were cultured in a dynamic bioreactor and assessed by biochemical assay and staining. Elastin demonstrated a better efficacy for recruiting cryopreserved chondrocytes onto the pore surface of constructs than poly-L-lysine. However, surface elastin and poly-L-lysine did not significantly enhance the biocompatibility to cryopreserved chondrocytes. Chondrocytes multiplied from cryopreserved chondrocytes in elastin-grafted constructs is faster than that in poly-L-lysine-grafted constructs. In addition, elastin could stimulate cryopreserved chondrocytes to synthesize more glycosaminoglycans and collagen than poly-L-lysine. Porous biomaterials with surface elastin and poly-L-lysine can maintain active chondrocytic proliferation and extracellular matrix secretion from chondrocytes with appropriate cryopreservation.

Application of albumin-grafted scaffolds to promote neocartilage formation.

This study investigates the capacity of albumin-grafted biomaterials as tissue engineering scaffolds to regenerate cartilaginous components. Porcine knee chondrocytes were seeded and cultivated in porous ternary matrix consisting of polyethylene oxide, chitin, and chitosan with surface albumin. The results revealed that the quantity of albumin did not affect the viability of porcine knee chondrocytes in the constructs. However, a high grafting concentration of albumin favored the adhesion of porcine knee chondrocytes on the scaffolding pore surface. After cultivation over 4 weeks, an increase in the concentration of albumin enhanced the quantities of porcine knee chondrocytes, glycosaminoglycans, and collagen in the constructs. The histological staining of porcine knee chondrocytes showed an active chondrocytic growth in the albumin-grafted constructs. In addition, the safranin-O staining indicated that the surface albumin could stabilize the secretion of glycosaminoglycans. Moreover, the immunochemical staining against type II collagen exhibited a regular production of collagen by phenotypic porcine knee chondrocytes in the constructs. Albumin-grafted polyethylene oxide/chitin/chitosan scaffolds can be a promising biomimetic substrate in neocartilage formation.

Integration of fiber optic-particle plasmon resonance biosensor with microfluidic chip.

This article reports the integration of the fiber optic-particle plasmon resonance (FO-PPR) biosensor with a microfluidic chip to reduce response time and improve detection limit. The microfluidic chip made of poly(methyl methacrylate) had a flow-channel of dimensions 4.0 cm × 900 µm × 900 µm. A partially unclad optical fiber with gold or silver nanoparticles on the core surface was placed within the flow-channel, where the volume of the flow space was about 14 µL. Results using sucrose solutions of various refractive indexes show that the refractive index resolution improves by 2.4-fold in the microfluidic system. The microfluidic chip is capable of delivering a precise amount of biological samples to the detection area without sample dilution. Several receptor/analyte pairs were chosen to examine the biosensing capability of the integrated platform: biotin/streptavidin, biotin/anti-biotin, DNP/anti-DNP, OVA/anti-OVA, and anti-MMP-3/MMP-3. Results show that the response time to achieve equilibrium can be shortened from several thousand seconds in a conventional liquid cell to several hundred seconds in a microfluidic flow-cell. In addition, the detection limit also improves by about one order of magnitude. Furthermore, the normalization by using the relative change of transmission response as the sensor output alleviate the demand on precise optical alignment, resulting in reasonably good chip-to-chip measurement reproducibility.

Using ac-field-induced electro-osmosis to accelerate biomolecular binding in fiber-optic sensing chips with microstructures.

This article reports the use of ac-field-induced charges at the corners of microstructures on fiber-optic sensing chips to generate electro-osmotic vortex flows in flow cell channels that can accelerate solute binding on the fiber. The sensing chip made of a cyclic olefin copolymer COC substrate contained a flow cell channel of dimensions 15 mm x 1 mm x 1 mm. A partially unclad optical fiber was placed within the channel. Relief-like strip structures of 25-mum thickness fabricated on the channel bottom were produced with an injection-molding process. The external electric field lines penetrating through the corners of the plastic microstructures induce charges on the corner surfaces to build up electrical double layers. When a high-frequency ac field (approximately 100 kHz) is used to flip the field polarities quickly, neutralization of the induced charge cannot be accomplished. The electrical double layer is therefore sustained. When absorbed charges in the double layer are driven by the external field, electro-osmotic flows are generated. The unclad portion of the fiber was coated with biotin-functionalized gold nanoparticles. The streptavidin solution was filled in the channel from the feeding tube, and the ac field (approximately 50 V/cm) was subsequently turned on for 30 s. The ac-field-induced electro-osmotic flows can accelerate solute transport in the sensing channel to enhance the binding kinetics of streptavidin molecules with biotin probes implanted on the gold nanoparticle surface. As a result, the fiber-optic localized plasmon resonance (FO-LPR) sensing signal becomes steady as soon as the external field is turned off. In contrast, the signal cannot reach steady state until 200-300 s in a typical static sensing cell. A significant reduction in the sensing response time is demonstrated. The binding assay of streptavidin with immobilized biotin on gold nanoparticle-coated sensing fibers was validated using this mixing device. The detection limit for streptavidin of approximately 10(-11) M is close to the reported values obtained using static cells. Similarly, the sensing response time of an orchid Odontoglossum ringspot virus (ORSV) sample was reduced from 1000 to 330 s when an external field was applied to mix the fluid for 60 s, even though the detection limit was maintained.

Study of cryopreservation of articular chondrocytes using the Taguchi method.

This study evaluates the effect of control factors on cryopreservation of articular cartilage chondrocytes using the Taguchi method. Freeze-thaw experiments based on the L(8)(2(7)) two-level orthogonal array of the Taguchi method are conducted, and ANOVA (analysis of variables) is adopted to determine the statistically significant control factors that affect the viability of the cell. Results show that the type of cryoprotectant, freezing rate, thawing rate, and concentration of cryoprotectant (listed in the order of influence) are the statistically significant control factors that affect the post-thaw viability. The end temperature and durations of the first and second stages of freezing do not affect the post-thaw viability. Within the ranges of the control factors studied in this work, the optimal test condition is found to be a freezing rate of 0.61+/-0.03 degrees C/min, a thawing rate of 126.84+/-5.57 degrees C/min, Me(2)SO cryoprotectant, and a cryoprotectant concentration of 10% (v/v) for maximum cell viability. In addition, this study also explores the effect of cryopreservation on the expression of type II collagen using immunocytochemical staining and digital image processing. The results show that the ability of cryopreserved chondrocytes to express type II collagen is reduced within the first five days of monolayer culture.

Hydrodynamic focusing investigation in a micro-flow cytometer.

Hydrodynamic focusing behavior is characterized by two fluids coflowing at different velocities inside a micro-flow cytometer. In this study, a two-fluid model has been established to describe the flow transport behavior and interaction of sample and sheath fluids. The analysis treats the sample and sheath fluids as two-dimensional, laminar, incompressible, and isothermal. The theoretical model comprises two groups of transient conservation equations of mass and momentum with consideration of the interfacial momentum exchange. The governing equations are solved numerically through an iterative SIMPLEC algorithm to determine the flow properties. Since the ratio of the sheath velocity to the sample velocity varies from 5 to 70, the predicted focusing width and length are in good agreement with the experimental data in the literature. In addition, the present study explored the hydrodynamic focusing flowfield as well as the pressure drop across a micro-flow cytometer and the time needed for the completion of one focusing event in detail. To enhance the understanding of hydrodynamic focusing in the design of cytometers, ten numerical experiments were conducted to examine the effects of the inner nozzle length, inner nozzle exit width, inner nozzle shape, and fluid properties on the width of the focused sample stream.