Dielectrophoresis System for Testing Antimicrobial Susceptibility of Gram-Negative Bacteria to ß-Lactam Antibiotics.

Gram-negative bacteria (GNBs) are common pathogens causing severe sepsis. Rapid evaluation of drug susceptibility would guide effective antibiotic treatment and promote life-saving. A total of 78 clinical isolates of 13 Gram-negative species collected between April 2013 and November 2013 from two medical centers in Tainan were tested. Bacterial morphology changes in different concentrations of antibiotics were observed under the electric field of a quadruple electrode array using light microscopy. The minimal inhibitory concentrations (MICs) of four antimicrobial agents, namely, cefazolin, ceftazidime, cefepime, and doripenem, were determined by the dielectrophoretic antimicrobial susceptibility testing (dAST) and by the conventional broth dilution testing (BDT). The antibiotics at the concentration of 1× MIC induced obvious morphological changes in susceptible GNBs, including cell elongation, cell swelling, or lysis, at 90 min. In contrast, resistant strains remained unchanged. The MIC results measured by dAST were in good agreement with those of BDT (essential agreement 95.6%). The category agreement rate was 89.2%, and the very major errors rate for dAST was 2.9%. In conclusion, dAST could accurately determine drug susceptibility within 90 min. Comprehensive tests by dAST for more drugs against more GNB species are possible in the future.

The ER Ca²âº sensor STIM1 regulates actomyosin contractility of migratory cells.

Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca(2+) sensor that triggers the store-operated Ca(2+) entry (SOCE). The clinical relevance of STIM1 has been highlighted in breast and cervical cancer, but the molecular mechanism by which STIM1 promotes cancer progression remains unclear. This study explores the regulatory mechanisms by which STIM1-dependent Ca(2+) signaling controls cancer cell migration. Three different SOCE inhibitors, SKF96365, 2-APB and YM-58483, significantly inhibited cervical cancer cell migration to a similar extent to that of STIM1 silencing. In contrast, STIM1 overexpression significantly enhanced cervical cancer cell migration. Live cell confocal images and three-dimensional tomograms showed that STIM1 formed aggregates and translocated towards the plasma membranes of migratory cells, and this was accompanied by increasing cytosolic Ca(2+) spikes. STIM1 silencing also inhibited the recruitment and association of active focal adhesion kinase (pTyr397-FAK) and talin at focal adhesions, indicating the blockade of force transduction from integrin signaling. Epidermal growth factor-induced phosphorylation of myosin II regulatory light chains was abolished by STIM1 knockdown and SOCE inhibition. Dual immunostaining of activated myosin II (pSer19-MLC) and actin revealed that actomyosin formation depended on STIM1-mediated Ca(2+) entry. Most importantly, STIM1 expression levels as well as SOCE activity controlled the generation of cell contractile force, as measured by the microfabricated post-array-detector system. These results highlight the unique role of STIM1-dependent Ca(2+) signaling in controlling cell migration by the regulation of actomyosin reorganization in conjunction with enhanced contractile forces.

Rapid detection of dermatophytes and Candida albicans in onychomycosis specimens by an oligonucleotide array.

BACKGROUND: Onychomycosis is a fungal infection of nails, leading to the gradual destruction of the nail plate. Treatment of onychomycosis may need long-time oral antifungal therapy that can have potential side effects, thus accurate diagnosis of the disease before treatment is important. Culture for diagnosis of onychomycosis is time-consuming and has high false-negative rates. To expedite the diagnosis, an oligonucleotide array, based on hybridization between immobilized oligonucleotide probes and PCR products, for direct detection of dermatophytes and Candida albicans in clinical specimens was evaluated. METHODS: Species-specific oligonucleotide probes designed from the internal transcribed spacer (ITS) regions of the rRNA gene were immobilized on a nylon membrane. The assay procedures consisted of PCR amplification of the ITS using universal primers, followed by hybridization of the digoxigenin-labeled amplicons to probes on the array. Thirty two nail samples (29 patients) were analyzed by the array, and the results were compared with those obtained by culture. Array-positive but culture-negative samples were confirmed by cloning and re-sequencing of the amplified ITS and by reviewing patient's clinical data. The total recovery of culture and confirmed array-positive but culture-negative results was considered 100% and was used for performance evaluation of both methods. RESULTS: Concordant results were obtained in 21 samples (10 positives and 11 negatives) by both methods. Eleven samples were array-positive but culture-negative; among them, 9 samples were considered true positives after discrepant analysis. Comparing with culture, the array had significantly higher sensitivity [100% (95% CI 82.2% -100%) vs 52.6% (28.9% -75.5%), p <0.001] and negative predictive value [100% (71.3% -100%) vs 59.1% (36.4% -79.3%), p <0.05), while no significant differences were observed in specificity (84.6% vs 100%, p =0.48) and positive predictive value (90.5% vs 100%, p =1.0). The whole procedures of the array were about 24 h, whilst results from culture take 1 to 3 weeks. CONCLUSIONS: The array offers an accurate and rapid alternative to culture. Rapid diagnosis can expedite appropriate antifungal treatment of onychomycosis. However, the single site nature of this study conducted at a referral hospital invites caution.

Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis.

Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.

Physicochemical properties and osteogenic activity of radiopaque calcium silicate-gelatin cements.

The purpose of this study is to evaluate the physicochemical properties and in vitro osteogenic activity of radiopaque calcium silicate-gelatin cements. The radiopacity, setting time, working time, flow, diametral tensile strength, pH value, washout resistance and morphology of the cements with gelatin (0, 5 and 10% by weight) were measured, which compared to a popular endodontic material, ProRoot white-colored mineral trioxide aggregate (WMTA). The cell morphology, cell attachment and proliferation, alkaline phosphatase and osteocalcin levels on the cements were measured by culturing the specimens with dental pulp cells. The results indicated that the presence of gelatin significantly (P < 0.05) reduced radiopacity and diametral tensile strength and prolonged setting time. Nevertheless, the 5 wt% gelatin cement had a radiopacity (5.1 mm of Al thickness) higher than ISO 6876:2001 standards (3 mm of Al thickness). The setting time (33 min), working time (9 min) and flow value (17.4 mm) of the 5 wt% gelatin cement were significantly (P < 0.05) better than those of WMTA (corresponding 165, 6 min and 14.2 mm). The fresh WMTA completely degraded after soaking in a physiological solution for 1 h, while the gelatin cements resisted washout, showing no noticeable breakdown even after 1 day of soaking. The gelatin cement enhanced the higher expression of cell attachment, proliferation and differentiation as compared to WMTA. It was concluded that the 5 wt% gelatin-calcium silicate hybrid cement appears to be promising as a radiopaque biomaterial for medical applications such as endodontics and vertebroplasty.

A point-of-care electrochemical biosensor for the rapid and sensitive detection of biomarkers in murine models with LPS-induced sepsis.

Sepsis is a life-threatening condition, which is irreversible if diagnosis and intervention are delayed. The response of the immune cells towards an infection triggers widespread inflammation through the production of cytokines, which may result in multiple organ dysfunction and eventual death. Conventional detection techniques fail to provide a rapid diagnosis because of their limited sensitivity and tedious protocol. This study proposes a point-of-care (POC) electrochemical biosensor that overcomes the limitations of current biosensing technologies in the clinical setting by its integration with electrokinetics, enhancing the sensitivity to picogram level compared with the nanogram limit of current diagnostic technologies. This biosensor promotes the use of a microelectrode strip to address the limitations of conventional photolithographic fabrication methods. Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and microRNA-155 (miR-155) were monitored in a lipopolysaccharide (LPS)-induced septic mouse model. The optimum target hybridization time in a high conductivity medium was observed to be 60 s leading to the completion of the whole operation within 5 min compared with the 4-h detection time of the traditional enzyme-linked immunosorbent assay (ELISA). The limit of detection (LOD) was calculated to be 0.84, 0.18, and 0.0014 pg mL-1, respectively. This novel sensor may have potential for the early diagnosis of sepsis in the clinical setting.

A rapid electrochemical biosensor based on an AC electrokinetics enhanced immuno-reaction.

Fluorescent labelling and chromogenic reactions that are commonly used in conventional immunoassays typically utilize diffusion dominated transport of analytes, which is limited by slow reaction rates and long detection times. By integrating alternating current (AC) electrokinetics and electrochemical impedance spectroscopy (EIS), we construct an immunochip for rapid, sensitive, and label-free detection. AC electroosmosis (ACEO) and positive dielectrophoresis (DEP), induced by a biased AC electric field, can rapidly convect and trap the analyte onto an EIS working electrode within a few minutes. This allows the change of electron-transfer resistance (ΔRet) caused by the antibody-antigen (IgG-protein A) binding to be measured and quantified in real time. The measured impedance change achieves a plateau after electrokinetic concentration for only 90 s, and the detection limit is able to reach 200 pg ml⁻¹. Compared to the conventional incubation method, the electrokinetics-enhanced method is approximately 100 times faster in its reaction time, and the detection limit is reduced by 30 times. The ΔRet of the positive response is two orders of magnitude higher than the negative control, demonstrating excellent specificity for practical applications.

Biopolymer coated gold nanocrystals prepared using the green chemistry approach and their shape-dependent catalytic and surface-enhanced Raman scattering properties.

This study deals with the preparation of multi-shaped nanoscale gold crystals under synthetically simple, green, and efficient conditions using a seed-mediated growth approach in the presence of hyaluronic acid (HA). These highly biocompatible multi-shaped gold nanocrystals were examined to evaluate their catalytic and surface enhanced Raman scattering (SERS) properties. The results show that the size and shape of the nanocrystals are mainly correlated to the amount of seed, seed size, HA concentration, and reaction temperature. Gold seeds accelerate the reduction of the gold precursor to form gold nanocrystals using HA. The HA serves as a reducing agent and a growth template for the reduction of Au(III) and nanocrystal stabilization. The multi-shaped gold nanocrystals showed superior catalytic properties and higher SERS performance. The simple, green approach efficiently controls the nanocrystals and creates many opportunities for future applications.

Voltage-modulated surface plasmon resonance biosensors integrated with gold nanohole arrays.

Surface plasmon resonance (SPR) has emerged as one of the most efficient and attractive techniques for optical sensors in biological applications. The traditional approach of an EC (electrochemical)-SPR biosensor to generate SPR is by adopting a prism underneath the sensing substrate, and an angular scan is performed to characterize the reflectivity of target analytes. In this paper, we designed and investigated a novel optical biosensor based on a hybrid plasmonic and electrochemical phenomenon. The SPR was generated from a thin layer of gold nanohole array on a glass substrate. Using C-Reactive Protein (CRP) as the target analyte, we tested our device for different concentrations and observed the optical response under various voltage bias conditions. We observed that SPR response is concentration-dependent and can be modulated by varying DC voltages or AC bias frequencies. For CRP concentrations ranging from 1 to 1000 µg/mL, at the applied voltage of -600 mV, we obtained a limit of detection for this device of 16.5 ng/mL at the resonance peak wavelength of 690 nm. The phenomenon is due to spatial re-distribution of electron concentration at the metal-solution interface. The results suggest that CRP concentration can be determined from the SPR peak wavelength shift by scanning the voltages. The proposed new sensor structure is permissible for various future optoelectronic integration for plasmonic and electrochemical sensing.

Screening of antibiotic susceptibility to ß-lactam-induced elongation of Gram-negative bacteria based on dielectrophoresis.

We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the ß-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 µm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to ß-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria.

A highly sensitive method for molecular diagnosis of fungal keratitis: a dot hybridization assay.

PURPOSE: Fungal keratitis (FK) is an important cause of ocular morbidity, especially for people living in the agricultural communities of the developing world. Current diagnostic methods may lack sensitivity (direct microscopy) or are time consuming (culture). The aim of this study was to develop a dot hybridization assay for sensitive and rapid diagnosis of FK. DESIGN: Evaluation of diagnostic test or technology. PARTICIPANTS AND CONTROLS: Fifty corneal scrapes (49 patients) from consecutive cases of clinically suspected microbial keratitis were analyzed prospectively. METHODS: Molecular detection of fungi in the scrapes was performed by amplification of the internal transcribed spacer region (ITS) that contained the target gene (5.8S rRNA gene) by polymerase chain reaction (PCR), followed by hybridization of the PCR product to a fungus-specific oligonucleotide probe immobilized on a nylon membrane. The results were compared with those obtained by gram-stain microscopy, culture, and gel electrophoresis of the PCR products. Discrepant results were resolved by cloning and resequencing of the amplified ITS fragments. MAIN OUTCOME MEASURES: Performance of the dot hybridization assay, including sensitivity, specificity, and positive and negative predictive values, was evaluated. RESULTS: Ten scrapes demonstrated positive results by both the dot hybridization assay and culture. However, 11 scrapes demonstrated positive results by the dot hybridization assay, but demonstrated negative results by culture, and 10 of the 11 samples were considered to be positive for FK by cloning and resequencing of the amplified ITS fragment and by a pathologic examination or clinical course review. The sensitivities for diagnosis of FK by the dot hybridization assay and culture were 100% and 50%, respectively, whereas the specificities were 96.7% and 100%, respectively. CONCLUSIONS: The dot hybridization assay is a highly sensitive and specific diagnostic tool for FK. The method provides a much higher sensitivity than that of culture (100% vs. 50%; P<0.001). The hybridization procedure can be finished within a working day. It is expected that the method can have an impact on the diagnosis and treatment of FK in the future. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Electroosmotic flow control in microfluidic chips using a self-assembled monolayer as the insulator of a flow field-effect transistor.

A novel concept for electroosmotic flow (EOF) control in a microfluidic chip is presented by using a self-assembled monolayer as the insulator of a flow field-effect transistor. Bidirectional EOF control with mobility values of 3.4 × 10(-4) and -3.1 × 10(-4) cm(2)/V s can be attained, corresponding to the applied gate voltage at -0.8 and 0.8 V, respectively, without the addition of buffer additives. A relatively high control factor (approximately 400 × 10(-6) cm(2)/V(2) s) can be obtained. The method presented in this study offers a simple strategy to control the EOF.

An AC electrokinetics-based electrochemical aptasensor for the rapid detection of microRNA-155.

Traditional immunosensors are often limited by low sensitivity and long detection times, for they usually depend on passive diffusion-dominated transport of target analytes for the binding reaction with a bio-recognition element such as enzymes, antibodies, and aptamers. Numerous studies rely on electric field manipulation by using alternating current (AC) electrokinetics to enhance the hybridization rate and reduce the hybridization time for faster and more efficient detection. This study demonstrated a rapid electrochemical aptasensor integrated with an AC electroosmotic (ACEO) flow phenomenon for the enhanced target hybridization of microRNA-155 (miR-155). Optimization of the electrokinetic conditions for target collection resulted in a saturation point after 75 s miR-155 was detected within the range of 1 aM-10 pM with a detection limit of 1 aM, which is 100 times lower and about 50 times faster compared with the conventional diffusion-dependent detection done for 1 h. The detection was also done in spiked serum samples, and a concentration range within the required detection range was obtained. The highly sensitive and specific results allow for the rapid and real-time sensing of target biomarkers, which can be used for the early detection of infection.

A rapid field-use assay for mismatch number and location of hybridized DNAs.

Molecular dielectrophoresis (DEP) is employed to rapidly (

Promoting effect of Antrodia camphorata as an immunomodulating adjuvant on the antitumor efficacy of HER-2/neu DNA vaccine.

It is well known that DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. Antrodia camphorata (AC) is a unique basidiomycete fungus of the Polyporaceae family that only grows on the aromatic tree Cinnamomum kanehirai Hayata (Lauraceae) endemic to Taiwan. Importantly, AC has been shown to be highly beneficial in the treatment and prevention of cancer. The goal of this study was to investigate whether AC is able to augment the antitumor immune properties of a HER-2/neu DNA vaccine in a mouse model in which p185neu is overexpressed in MBT-2 tumor cells. Compared with the mice that received the HER-2/neu DNA vaccine alone, co-treatment with AC suppressed tumor growth and extended the survival rate. This increase in the antitumor efficacy was attributed to the enhancement of the Th1-like cellular immune response by the HER-2/neu DNA vaccine-AC combination. Evidence for this came from the marked increase in the IFN-gamma mRNA expression in CD4+ T cells in the draining inguinal lymph nodes, an increase in the number of functional HER-2/neu-specific CTLs, and the increased tumor infiltration of both CD4+ and CD8+ T cells, depletion of which abolishes the antitumor effect of the HER-2/neu DNA vaccine-AC therapy. Our results further indicate that the treatment of mice with AC enhanced DC activation and production of Th1-activating cytokines (e.g. IL-12, and IFN-alpha) in the draining lymph nodes, which were sufficient to directly stimulate T cell proliferation and higher IFN-gamma production in response to ErbB2. Overall, these results clearly demonstrate that AC represents a promising immunomodulatory adjuvant that could enhance the therapeutic potency of HER-2/neu DNA vaccines in cancer therapy.

A continuous high-throughput bioparticle sorter based on 3D traveling-wave dielectrophoresis.

We present a high throughput (maximum flow rate approximately 10 microl/min or linear velocity approximately 3 mm/s) continuous bio-particle sorter based on 3D traveling-wave dielectrophoresis (twDEP) at an optimum AC frequency of 500 kHz. The high throughput sorting is achieved with a sustained twDEP particle force normal to the continuous through-flow, which is applied over the entire chip by a single 3D electrode array. The design allows continuous fractionation of micron-sized particles into different downstream sub-channels based on differences in their twDEP mobility on both sides of the cross-over. Conventional DEP is integrated upstream to focus the particles into a single levitated queue to allow twDEP sorting by mobility difference and to minimize sedimentation and field-induced lysis. The 3D electrode array design minimizes the offsetting effect of nDEP (negative DEP with particle force towards regions with weak fields) on twDEP such that both forces increase monotonically with voltage to further increase the throughput. Effective focusing and separation of red blood cells from debris-filled heterogeneous samples are demonstrated, as well as size-based separation of poly-dispersed liposome suspensions into two distinct bands at 2.3 to 4.6 microm and 1.5 to 2.7 microm, at the highest throughput recorded in hand-held chips of 6 microl/min.

Entrapment and measurement of a biologically functionalized microbead with a microwell electrode.

A chip with integrated electrophoretic and electrochemical systems was developed to manipulate either an individual microbead or a cell inside a microwell electrode (MWE) for electrochemical measurement. The optimal MWE geometry (30 microm diameter and 25 microm depth) was designed to accommodate the micro particles according to the simulated results. A chip device was sequentially built from a slide patterned with Pt electrodes, an adhesive tape defined with a flow channel (200 microm in width and 25 microm in height), and an indium tin oxide (ITO) cover. The MWE not only generated an active electrophoretic force to trap the particle but also provided a low flow velocity area (LFVA) to stabilize the trapped bead or cell in a continuous flow. Scanning electrochemical microscopy (SECM) theory was employed to explain the electrochemical behaviors of the MWE. An enhanced current was confirmed as the redox recycling effect on the conductive ITO cover. The catalytic reaction of an individual alkaline phosphatase coated microbead (ALP-bead) was electrochemically detected with the MWE after being trapped. The ALP on the trapped ALP-bead catalyzed the hydrolysis of p-aminophenylphosphate (PAPP) to p-aminophenol (PAP), and then a decaying amperogram (+0.3 V vs. Ag/AgCl) due to a tiny PAP quantity around the MWE was observed.

In situ study of EDC/NHS immobilization on gold surface based on attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS).

In this study, the time-dependent reaction between 11-mercaptoundecanoic acid (11-MUA) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) is precisely characterized using surface enhanced infrared absorption spectroscopy (SEIRAS). According to the high correlation between the spectral results of SEIRAS and the electrochemical behavior, it strongly demonstrates that the EDC/NHS reaction would be obviously interfered by phosphate ions in the neutral pH condition (pH = 7.0).

Simple In-House Fabrication of Microwells for Generating Uniform Hepatic Multicellular Cancer Aggregates and Discovering Novel Therapeutics.

Three-dimensional (3D) cell culture models have become powerful tools because they better simulate the in vivo pathophysiological microenvironment than traditional two-dimensional (2D) monolayer cultures. Tumor cells cultured in a 3D system as multicellular cancer aggregates (MCAs) recapitulate several critical in vivo characteristics that enable the study of biological functions and drug discovery. The microwell, in particular, has emerged as a revolutionary technology in the generation of MCAs as it provides geometrically defined microstructures for culturing size-controlled MCAs amenable for various downstream functional assays. This paper presents a simple and economical microwell fabrication methodology that can be conveniently incorporated into a conventional laboratory setting and used for the discovery of therapeutic interventions for liver cancer. The microwells were 400-700 µm in diameter, and hepatic MCAs (Huh-7 cells) were cultured in them for up to 5 days, over which time they grew to 250-520 µm with good viability and shape. The integrability of the microwell fabrication with a high-throughput workflow was demonstrated using a standard 96-well plate for proof-of-concept drug screening. The IC50 of doxorubicin was determined to be 9.3 µM under 2D conditions and 42.8 µM under 3D conditions. The application of photothermal treatment was demonstrated by optimizing concanavalin A-FITC conjugated silica-carbon hollow spheres (SCHSs) at a concentration of 500:200 µg/mL after a 2 h incubation to best bind with MCAs. Based on this concentration, which was appropriate for further photothermal treatment, the relative cell viability was assessed through exposure to a 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of using photothermal treatment as a potential therapeutic intervention. The proposed microwell integration is envisioned to serve as a simple in-house technique for the generation of MCAs useful for discovering therapeutic modalities for liver cancer treatment.

A new protein A assay based on Raman reporter labeled immunogold nanoparticles.

A unique, sensitive, highly specific, and photobleaching-resistant immunoassay system utilizing gold nanoparticles and surface-enhanced Raman scattering (SERS) is described. This new system, featuring a capability of bifunctional analysis, is manufactured by chemisorption of antibody immunoglobulin G (IgG) on gold nanoparticles (AuNP), followed by coupling the Raman-active reporter molecule, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to the surface of IgG-AuNP. The adsorbed DTNB molecules exhibit strong Raman signals via both electromagnetic and chemical enhancement. The narrow spectral widths and high photostability assure the system to be an excellent detection label. This SERS-based immunoassay is applied to the detection of protein A, which is a specific surface antigen of Staphylococcus aureus. A working curve is obtained by plotting the intensity of the SERS signal of symmetric NO(2) stretching of DTNB at 1,333 cm(-1) versus the concentration of the analyte (antigen). A dynamic range of two to three orders of magnitude and a detection limit of 1 pg/mL of protein A are achieved.