Clinical Application of Surface Plasmon Resonance-Based Biosensors for Fetal Fibronectin Detection

Preterm birth is the leading cause of perinatal morbidity and mortality. Fetal fibronectin (fFN), a glycoprotein in the extracellular matrix of the amniotic membranes, is the most powerful biomarker for predicting the risk of preterm birth. Biosensors using the surface plasmon resonance (SPR) response are potentially useful in quantitatively measuring molecules. We established a standard calibration curve of SPR intensity against fFN concentration and used the SPR-based biosensor to detect fFN concentrations in the cervicovaginal secretions of pregnant women between 22 and 34 weeks of gestation. The calibration curve extends from 0.5 ng/mL to 100 ng/mL with an excellent correlation (R2 = 0.985) based on standard fFN samples. A cutoff value of 50 ng/mL fFN concentration in commercial ELISA kits corresponds to a relative intensity of 17 arbitrary units (a.u.) in SPR. Thirty-two pregnant women were analyzed in our study. In 11 women, the SPR relative intensity was greater than or equal to 17 a.u., and in 21 women, the SPR relative intensity was less than 17 a.u. There were significant differences between the two groups in regular uterine contractions (p = 0.040), hospitalization for tocolysis (p = 0.049), and delivery weeks (p = 0.043). Our prospective study concluded that SPR-based biosensors can quantitatively measure fFN concentrations. These results reveal the potential utility of SPR-based biosensors in predicting the risk of preterm birth

Development of a Surface Plasmon Resonance Biosensor for Real-Time Detection of Osteogenic Differentiation in Live Mesenchymal Stem Cells

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis

Anti Human Fibronectin–Gold Nanoparticle Complex, a Potential Nanobiosensor Tool for Detection of Fibronectin in ECM of Cultured Cells

Specific protein detection by means of antibody-nanoparticle conjugates is a new field in medical nanobiotechnology. Among many nanoparticles used, gold nanoparticles show strong light-absorption properties which have been exploited in designing nanobiosensors. Fibronectin (FN) plays an important role in extracellular matrix (ECM) structure and function of normal cells; however, in conditions like lung carcinoma, its expression increases, especially in non small cell lung carcinoma (NSCLC). In this study, we conjugated gold nanoparticles to human fibronectin antibody (anti-hFN) to design a colorimetric nanobiosensor for detection of FN present in ECM of cultured cells. Three different cell lines, namely A549 (target cells), AGO-1522 (control cells), and Nalm-6 (negative control cells), were used to compare changes in color resulting from aggregation of gold nanoparticles due to higher amount of FN. Our construct was able to detect increased level of FN which was distinguishable visually by change in color and could be confirmed by spectrophotometer as well. © 2014, Springer Science+Business Media New York

Cell Culture on MEMS Platforms: A Review

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bioincompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bioincompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented.Institute of Bioengineering and Nanotechnology (Singapore)Singapore. Biomedical Research CouncilSingapore. Agency for Science, Technology and ResearchSingapore. Agency for Science, Technology and Research (R-185-001-045-305)Singapore. Ministry of EducationSingapore. Ministry of Education (Grant R-185- 000-135-112)Singapore. National Medical Research CouncilSingapore. National Medical Research Council (Grant R-185-000-099-213)Jassen Cilag (Firm)Singapore-MIT Alliance (Computational and Systems Biology Flagship Project)Global Enterprise for Micro-Mechanics and Molecular Medicin

Biosensors in clinical practice : focus on oncohematology

Biosensors are devices that are capable of detecting specific biological analytes and converting their presence or concentration into some electrical, thermal, optical or other signal that can be easily analysed. The first biosensor was designed by Clark and Lyons in 1962 as a means of measuring glucose. Since then, much progress has been made and the applications of biosensors are today potentially boundless. This review is limited to their clinical applications, particularly in the field of oncohematology. Biosensors have recently been developed in order to improve the diagnosis and treatment of patients affected by hematological malignancies, such as the biosensor for assessing the in vitro pre-treatment efficacy of cytarabine in acute myeloid leukemia, and the fluorescence resonance energy transfer-based biosensor for assessing the efficacy of imatinib in chronic myeloid leukemia. The review also considers the challenges and future perspectives of biosensors in clinical practice

Perinteiset ja uudet menetelmät, joilla tutkitaan lääkeaineen solukalvon läpäisevyyttä sekä lääkeaineen ja solujen välisiä vuorovaikutuksia lääkekehityksen varhaisissa vaiheissa (kirjallisuuskatsaus) : Lääkeaineen ja solujen välisten vuorovaikutusten tutkiminen pintaplasmoniresonanssi-tekniikalla (erikoistyö)

There is a strong need for new in vitro methods in early drug development that predict in vivo conditions more reliably. One of the prerequisites for successful drug therapy is sufficient permeability. A drug needs to be transported through a cell membrane before it can have a pharmacological effect. Therefore, the drug-cell interactions are studied in the early stage of the drug development process. The literature review of this work covers the traditional in vitro and in silico methods of predicting the permeability of drugs across the intestinal membrane. The widely applied methods are reviewed briefly and the predictability of the methods is evaluated. Moreover, the surface plasmon resonance (SPR) technique is introduced. The principle of SPR and its applications for predicting intestinal permeability using lipid membranes resembling the intestinal membrane and for studying drug-cell interactions are discussed. The advantage of the SPR technique is that it is an optical method which allows real-time monitoring under a constant flow without labeling agents. The aim of the experimental part of this work was to evaluate the suitability of the SPR technique for cell-based studies to monitor drug-cell interactions in native cellular environments. Previously, the SPR technique has been almost merely used in routine biomolecular interaction analysis. Recently, the SPR technique has also been applied to cellbased assays but in those studies the reason for the SPR signal responses is generally poorly discussed. The objective of the experimental study was to evaluate and optimize different cell culturing approaches for living cell sensing for SPR, i.e. cells immobilized on the roof of the PDMS molded flow channel in the SPR instrument and cells immobilized directly on the SPR sensor surface. ARPE-19 cells were immobilized on the PDMS substrates but the challenge of imaging cell monolayers on PDMS molded SPR flow channels suggested that immobilizing the cells directly on the SPR sensor surface would be a more straightforward procedure. Hence, ARPE-19 and MDCKII cell culturing protocols were optimized for successful immobilization of confluent cell monolayers directly on the SPR sensor surface. However, ARPE-19 cells showed poor resistance against shear stress in the flow channel; whereas MDCKII cells showed much better resistance against shear stress in the flow channel. Therefore, only MDCKII cells immobilized on the SPR sensor surfaces were used for drug-cell interaction studies. After three days of culture MDCKII cells were exposed to test compounds in separate SPR measurements. The used test compounds were propranolol, D-mannitol, D-glucose and HSPC:Chol liposomes. During the SPR measurements, the changes in the SPR peak minimum angular position and SPR peak minimum intensity were recorded in real-time, and these were further used for analysis after the measurements. The results showed that clear differences in both SPR signals between propranolol and D-mannitol were observed when the cells were exposed to the test compounds. Propranolol diffuses effectively by the transcellular pathway into cells whereas D-mannitol uses the paracellular pathway. This indicates that the introduced SPR approach may be a potential in vitro method in order to provide real-time information on the permeability of drugs and possibly on cell uptake mechanisms of nanoparticles for a better mechanistic understanding of drug-cell interactions on a cellular level.Lääkekehityksen varhaisiin vaiheisiin tarvitaan uusia, luotettavammin in vivo -tilannetta ennustavia in vitro -tutkimusmenetelmiä. Lääkeaineen täytyy kulkeutua solukalvon läpi ennen kuin se voi päästä vaikutuskohtaansa. Tämän vuoksi lääkeaineen vuorovaikutusta solujen kanssa on tärkeää tutkia lääkekehitysen mahdollisimman varhaisessa vaiheessa. Kirjallisuuskatsauksessa tarkastellaan perinteisiä in vitro ja in silico -menetelmiä, joita käytetään lääkeaineen imeytymisen ennustamiseen ruoansulatuskanavasta. Katsauksessa kuvataan lyhyesti yleisimmin käytettyjä tutkimusmenetelmiä sekä arvioidaan menetelmien luotettavuutta erityisesti lääkeaineen in vivo -imeytymisen ennustamisessa. Lisäksi katsauksessa esitellään uusi pintaplasmoniresonanssi (SPR)-menetelmä ennustamaan lääkeaineen imeytymistä solukalvon läpi. SPR-menetelmää on käytetty muun muassa lääkeaineen imeytymisen tutkimisessa keinotekoisten lipidikerrosten avulla sekä lääkeaineen ja solujen välisten vuorovaikutusten tutkimisessa SPR-sensorin pinnalla. SPR-menetelmän etuna on, että sillä on mahdollista tutkia lääkeaineen ja solujen välisiä vuorovaikutuksia reaaliaikaisesti, jatkuvassa virtauksessa ja ilman merkkiaineita. Erikoistyön tavoitteena oli selvittää soveltuuko SPR-menetelmä lääkeaineen ja solujen välisten vuorovaikutusten tutkimiseen. SPR-menetelmää on käytetty aiemmin lähinnä biokemiallisissa tutkimuksissa tarkasteltaessa molekyylien välisiä vuorovaikutuksia. Menetelmällä on tehty myös solututkimuksia, mutta niissä on analysoitu yksinkertaistetusti mitattuja signaalin muutoksia. Erikoistyön tavoitteena oli löytää optimoidut olosuhteet, joissa voitaisiin tehdä SPR-solumittauksia. Tavoitteena oli kasvattaa soluja SPR-laitteen PDMS:lla päällystetyn virtauskanaviston pinnalla tai suoraan SPR-sensorin pinnalla. ARPE-19-solut onnistuttiin kasvattamaan yhtenäisenä solukerroksena PDMS:n pinnalla, mutta solukerroksen tarkastelu PDMS-virtauskanaviston pinnalla oli haastavaa. Tämän vuoksi solut päätettiin kasvattaa SPR-sensorin pinnalla. Viljelyolosuhteet optimoitiin siten, että ARPE-19 ja MDCKII-solut kasvoivat yhtenäisenä solukerroksena SPR-sensorilla. Virtausmittauksissa kuitenkin selvisi, että ARPE-19-solut eivät kestäneet puskurin virtauksen aiheuttamia olosuhteita virtauskanavistossa, kun taas MDCKII-solut kestivät niitä hyvin. Tämän vuoksi varsinaiset SPR-mittaukset tehtiin vain MDCKII-soluilla. Kun MDCKII-soluja oli viljelty kolme päivää SPR-sensorin pinnalla, solukerroksen ja tutkimusaineiden välinen vuorovaikutus mitattiin SPR-laitteella erillisissä mittauksissa. Tutkimusaineina käytettiin propranololia, Dmannitolia, D-glukoosia ja HSPC:KOL-liposomeja. Mittauksissa tarkasteltiin SPRminimikulman ja SPR-piikin minimi-intensiteetin muutosta reaaliajassa, kun solukerros altistettiin tutkimusaineille. Tulokset osoittivat, että SPR-menetelmällä voitiin havaita selkeä eroavaisuus molemmissa SPR-signaaleissa propranololin ja D-mannitolin välillä. Propranololi läpäisee solukerroksen transsellulaarisesti, kun taas D-mannitoli käyttää parasellulaarista reittiä. Tämä tarkoittaa, että menetelmällä oli mahdollista erottaa nämä imeytymisreitit toisistaan. Tulokset osoittavat, että SPR-tekniikka voi olla potentiaalinen in vitro -menetelmä tutkimaan lääkeaineiden vuorovaikutuksia solukerroksen kanssa. Menetelmällä olisi mahdollista tutkia esimerkiksi lääkeaineaineiden ja nanopartikkelien imeytymistä reaaliaikaisesti ja ilman merkkiaineita

An Anti-Pyruvate Kinase Monoclonal Antibody And Translocated Intimin Receptor (tir) For Specific Detection Of Listeria Species And Shiga-Toxigenic Escherichia Coli

Foodborne illnesses pose a significant health concern and economic impact worldwide. In this study, we aimed at developing alternate and improved methods for Shiga toxigenic E. coli (STEC) and Listeria species detection. In Listeria monocytogenes, an auxiliary secretory system, SecA2, plays an important role in translocating virulence and housekeeping proteins to cell surface to aid bacteria to maintain saprophytic and intracellular life styles. Here we investigated if pyruvate kinase (PyK), present in both pathogenic and nonpathogenic Listeria, is translocated by SecA2 system and determined its potential application in immunologic detection of these bacteria. Additionally, cell surface localization and enzymatic activity of PyK were examined. Enzyme immunoassay with anti-PyK antibody, MAb EM-7H10, indicated the presence of PyK in all Listeria species except L. roquortiae. Immunofluorescence assay confirmed surface localization. Analysis of L. monocytogenes ΔsecA2 mutant revealed the absence of PyK in cell wall and the supernatant fractions along with reduced levels in the intracellular fraction indicating that PyK translocation to cell surface is SecA2-dependent. Reverse transcriptase PCR (RT-PCR) confirmed reduced levels of PyK transcript in the ΔsecA2 mutant indicating SecA2-dependent regulation of pyk. Furthermore, PyK expression was found to be 10-fold higher in L. monocytogenes cultured in Brain-Heart Infusion Broth (BHI), Tryptic Soy Broth (TSB) and buffered Listeria enrichment broth (BLEB) than in University of Vermont medium (UVM) or Fraser Broth (FB). In summary, PyK is determined to be a SecA2-dependent surface displayed glycolytic enzyme present in both pathogenic and nonpathogenic Listeria, which could serve as a strong immunologic target for Listeria species detection. Shiga toxigenic E. coli (STEC) has been implicated in several foodborne outbreaks exhibiting severe hemolytic uremic syndrome (HUS) and fatalities. Here, we focus on a novel approach for STEC detection. Translocated Intimin Receptor (TIR) binds exclusively with intimin, a STEC adhesion protein which mediates intimate attachment of the bacteria to the host cell. This receptor-ligand system is unique to STEC and can be used for its detection on biosensor platforms. Collectively, data provide strong evidence for the use of anti-PyK antibody and TIR and for specific detection of Listeria species and STEC, respectively

Development of Dual Imaging Single Vesicle Technology for Exosome Characterization and Early Cancer Detection

Single vesicle molecular profiling has the potential to transform cancer detection and monitoring by precisely probing cancer-associated exosomes in the presence of normal exosomes in body fluids. However, it is challenging due to the small exosome size, low abundance of antigens on individual exosomes, and complex biological matrix. A facile dual imaging single vesicle technology (DISVT) has been developed and described for surface protein profiling of individual exosomes and quantification of target-specific exosome subtypes in biofluids by integrating multiple components, including direct molecular exosome capture from diluted biofluids, dual exosome-protein light scattering/fluorescence imaging, and fast Python-programing based image analysis. This technology uses strong surface plasmon light scattering properties of gold nanoparticles (AuNPs) to detect targeted surface protein markers with darkfield imaging on exosomes at the single particle level. Meanwhile, fluorescent imaging uses a fluorescent near-infrared dye to localize captured exosomes. A customized microscope achieved dual light scattering/fluorescence imaging. By optimizing various parameters, including concentration, binding time, and binding temperature of the AuNPs and dye, this DISVT can detect targeted surface protein markers of interest at the single vesicle level. Using DISVT, we characterized several protein markers on exosomes derived from several model breast cancer (BC) cells, validating them with standard ELISA. We demonstrated the potential of DISVT for early cancer detection using Human epidermal growth factor receptor 2 (HER2)-positive BC as the disease model by profiling HER2-positive exosomes in plasma from HER2-positive BC patients at multiple stages, comparing them with healthy donors and HER2-negative BC patients. We demonstrated that the DISVT, but not ELISA, detected BC at early stages. The DISVT differentiated HER2-positive- from HER2-negative- BC. The amount of tumor-associated exosomes showed to be tripled in locally advanced- compared to early-stage- patients. Our studies suggest that single exosome surface protein profiling with DISVT can provide a novel and highly sensitive method for early cancer detection and quantitative monitoring. Due to the advantages in simplicity, sample consumption, speed, and cost, this DISVT has the potential to accelerate the translation of exosomes into clinical use, leading to a new generation of liquid biopsy for detecting and monitoring cancer and other diseases