Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors detect Bacillus anthracis at 300 spores/mL

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 2.48 mm2 were fabricated. Antibody specific to Bacillus anthracis (BA, Sterne strain 7702) spores was immobilized on PEMC sensors, and exposed to spores (300 to 3x10(6) spores/mL). The resonant frequency decreased at a rate proportional to the spore concentration and reached a steady state frequency change of 5+/-5 Hz (n=3), 92+/-7 Hz (n=3), 500+/-10 Hz (n=3), 1030+/-10 Hz (n=2), and 2696+/-6 Hz (n=2) corresponding to 0, 3x10(2), 3x10(3), 3x10(4), and 3x10(6) spores/mL, respectively. The reduction in resonant frequency is proportional to the change in cantilever mass, and thus the observed changes are due to the attachment of spores on the sensor surface. Selectivity of the antibody-functionalized sensor was determined with samples of BA (3x10(6)/mL) mixed with Bacillus thuringiensis (BT; 1.5x10(9)/mL) in various volume ratios that yielded BA:BT ratios of 1:0, 1:125, 1:250, 1:500 and 0:1. The corresponding resonance frequency decreases were, respectively, 2345, 1980, 1310, 704 and 10 Hz. Sample containing 100% BT spores (1.5x10(9)/mL and no BA) gave a steady state frequency decrease of 10 Hz, which is within noise level of the sensor, indicating excellent selectivity. The observed binding rate constant for the pure BA and BT-containing samples ranged from 0.105 to 0.043 min-1 in the spore concentration range 300 to 3x10(6)/mL. These results show that detection of B. anthracis spore at a very low concentration (300 spores/mL) and with high selectivity in presence of another Bacillus spore (BT) can be accomplished using piezoelectric-excited millimeter-sized cantilever sensors.     

Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1 mL/min

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4mm(2) sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (+/-20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1-17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1h was 90+/-5 Hz (n=2), and 162+/-10 Hz (n=2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388+/-10 (n=2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.     

Detect of Escherichia coli O157:H7 in ground beef samples using piezoelectric excited millimeter-sized cantilever (PEMC) sensors

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 4 mm2 were fabricated. An antibody specific to Escherichia coli (anti-E. coli) O157:H7 was immobilized on PEMC sensors, and exposed to samples containing E. coli O157:H7 (EC) prepared in various matrices: (1) broth, broth plus raw ground beef, and broth plus sterile ground beef without inoculation of E. coli O157:H7 served as controls, (2) 100 mL of broth inoculated with 25 EC cells, (3) 100 mL of broth containing 25 g of raw ground beef and (4) 100 mL of broth with 25 g of sterile ground beef inoculated with 25 EC cells. The total resonant frequency change obtained for the broth plus EC samples were 16+/-2 Hz (n=2), 30 Hz (n=1), and 54+/-2 Hz (n=2) corresponding to 2, 4, and 6h growth at 37 degrees C, respectively. The response to the broth plus 25 g of sterile ground beef plus EC cells were 21+/-2 Hz (n=2), 37 Hz (n=1), and 70+/-2 Hz (n=2) corresponding to 2, 4, and 6 h, respectively. In all cases, the three different control samples yielded a frequency change of 0+/-2 Hz (n=6). The E. coli O157:H7 concentration in each broth and beef samples was determined by both plating and by pathogen modeling program. The results indicate that the PEMC sensor detects E. coli O157:H7 reliably at 50-100 cells/mL with a 3 mL sample.     

Sensitive and selective detection of mycoplasma in cell culture samples using cantilever sensors

In this article we report a new biosensor‐based method that is more sensitive and rapid than the current approach for detecting mycoplasma in cell culture samples. Piezoelectric‐excited millimeter‐sized cantilever (PEMC) sensors respond to mass change via resonant frequency change. They are sensitive at femtogram level and can be used directly in liquid for label‐free detection. Common cell culture contaminant, Acholeplasma laidlawii was detected in both buffer and cell culture medium. Two different sources (positive control from a commercial kit and ATCC 23206) were analyzed using antibody‐immobilized PEMC sensor. Resonant frequency decrease caused by binding of A. laidlawii was monitored in real‐time using an impedance analyzer. Positive detection was confirmed by a second antibody binding. The limit of detection (LOD) was lower than 10³ CFU/mL in cell culture medium using PEMC sensor while parallel ELISA assays showed LOD as 10⁷ CFU/mL. This study shows that PEMC sensor can be used for sensitive and rapid mycoplasma detection in cell culture samples. Biotechnol. Bioeng. 2010;105: 1069–1077. © 2009 Wiley Periodicals, Inc.     

Detection and quantification of proteins using self-excited PZT-glass millimeter-sized cantilever

A composite self-excited PZT-glass cantilever (4mm in length and 2mm wide) was fabricated and used to measure the binding and unbinding of model proteins. A key feature of the cantilever is that its resonant frequency is dependent on its mass. The fabricated cantilever has mass change sensitivity in liquid of 7.2 x 10(-11)g/Hz. Resonant frequency change was measured as protein reacted or bound with the sensing glass cantilever surface. Protein concentrations, 0.1 and 1.0mg/mL, which resulted in nanogram mass change were successfully detected. The mass change sensitivity gave a total mass change of 54+/-0.45 ng for the binding of anti-rabbit IgG (biotin conjugated) to rabbit IgG immobilized cantilever and the subsequent binding of captavidin. The unbinding of anti-rabbit IgG and captavidin gave a total mass change of 54+/-1.70 ng. Fluorescence based assays showed the combined mass of both proteins in the released samples was 54+/-2.24 ng. The binding kinetics of the model proteins is modeled as first order. The initial binding rate constant of anti-rabbit IgG to rabbit IgG was 1.36+/-0.02(min(mg/mL))(-1). The initial binding rate constant of captavidin to biotinylated anti-rabbit IgG was (2.57 x 10(-1))+/-0.003(min(mg/mL))(-1). The significance of the results we report here is that millimeter-sized PZT-actuated glass cantilevers have the sensitivity to measure in real-time protein-protein binding, and the binding rate constant.     

Micro-machined piezoelectric membrane-based immunosensor array

This paper reports a micro-machined piezoelectric membrane-based biosensor array for immunoassay. Goat immunoglobulin G (IgG) and HBsAg were immobilized as the probe molecules on the square piezoelectric membranes of the sensors that have dimensions of 3.5 microm x 500 microm x 500 microm. Due to the mass sensitive nature of these sensors, their resonant frequencies were depressed after the anti-goat IgG or anti-HBsAg was captured by the goat IgG or HBsAg. The resonant frequencies of the sensors were measured by an impedance analyzer. The experimental results demonstrate that the measured frequency change varies from 100 to 700 Hz, and the mass sensitivity of the device is estimated to be about 6.25 Hz/ng. A near linear relationship between the frequency change and the concentration of goat IgG was obtained, and the mass of the attached anti-goat IgG was calculated. The preliminary results discussed in this work indicate that the micro-machined piezoelectric membrane-based biosensor has a potential application as an immunosensor.     

Detection of pathogen Escherichia coli O157:H7 using self-excited PZT-glass microcantilevers

Composite self-excited PZT-glass cantilevers (5 and 3 mm in length, 1.8 and 2.0 mm wide) were fabricated and their resonance characteristics were determined in air and at 1 mm liquid immersion. In air, resonance occurred at 65.8 and 63.4 kHz for the two cantilevers used in this paper. Monoclonal antibody (MAb) specific to the pathogen Escherichia coli (E. coli) O157:H7 was immobilized at the cantilever glass tip, and then exposed to pathogen in the concentration range of 7x10(2) to 7x10(7)bacteria/mL. Resonance of the second mode decreased due to pathogen attachment in accordance with a proposed kinetic model. The specific attachment rate constant was found to be 3x10(-9) to 5x10(-9) min-1 (cell/mL)-1. Exposure to a mixed population containing both a pathogenic and non-pathogenic strain showed that the antibody-immobilized cantilever is highly selective, thus demonstrating its usefulness for detecting water-borne pathogens.     

Detection of pathogen Escherichia coli O157:H7 AT 70 cells/mL using antibody-immobilized biconical tapered fiber sensors

Optical fibers (core diameter 8 microm, cladding diameter 125 microm) was tapered to a waist diameter in the range of 8-12 microm, and then a monoclonal antibody to the pathogen, Escherichia coli O157:H7 was covalently bonded to the surface of the tapered region. Using 470 nm light, the taper was exposed to various concentrations (7 x 10(7), 7 x 10(5), 7 x 10(3), and 70 cells/mL) of the pathogen, and the sensor showed changes in transmitted light as the antigen attached to the antibody on the taper surface. The response was equal and opposite when the pathogen was released from the surface using a low pH buffer. The magnitude of the change was inversely proportional to the concentration of the pathogen. The sensor showed good sensitivity at as low a concentration as 70 cells/mL. The antibody-immobilized taper sensor was also exposed to a mixture of the pathogen and a non-pathogenic variant (JM101) at 0%, 50% and 70% by concentration. The sensor showed good selectivity to the pathogenic antigen. A first order attachment kinetic model is proposed to quantify the rate of attachment of pathogen to the sensor surface. The kinetic rate constant (k) of E. coli O157:H7 to the fiber was found to vary in the range of (2.5-6.1) x 10(-9) min(-1) (cells/mL)(-1).     

Near real-time detection of Cryptosporidium parvum oocyst by IgM-functionalized piezoelectric-excited millimeter-sized cantilever biosensor

Piezoelectric-excited millimeter-sized cantilever (PEMC) biosensors were fabricated and functionalized with immunoglobulin M (IgM) for the detection of Cryptosporidium parvum oocyst in a flow configuration at 1 mL/min. The detection of 100, 1000, and 10,000 oocysts/mL was achieved with a positive sensor response in less than 1 min. Bovine serum albumin (BSA) was used as a blocking agent in each experiment and was shown to eliminate non-specific binding. The sensor's resonance frequency response correlates with C. parvum oocyst concentration logarithmically. The oocyst attachment rate was found to increase by an order of magnitude in increasing concentration from 100 to 10,000 oocysts/mL. The significance of these results is that IgM-functionalized PEMC sensors are highly selective and sensitive to C. parvum oocyst and therefore, have the potential to accurately identify and quantify C. parvum oocyst in drinking water.     

The effect of salt and phage concentrations on the binding sensitivity of magnetoelastic biosensors for Bacillus anthracis detection

This article presents an investigation of the effect of salt and phage concentrations on the binding affinity of magnetoelastic (ME) biosensors. The sensors were fabricated by immobilizing filamentous phage on the ME platform surface for the detection of Bacillus anthracis spores. In response to the binding of spores to the phage on the ME biosensor, a corresponding decrease occurs in resonance frequency. Transmission electron microscopy (TEM) was used to verify the structure of phage under different combinations of salt/phage concentration. The chemistry of the phage solution alters phage bundling characteristics and, hence, influences both the sensitivity and detection limit of the ME biosensors. The frequency responses of the sensors were measured to determine the effects of salt concentration on the sensors' performance. Scanning electron microscopy (SEM) was used to confirm and quantify the binding of spores to the sensor surface. This showed that 420 mM salt at a phage concentration of 1 x 10(11) vir/mL results in an optimal distribution of immobilized phages on the sensor surface, consequently promoting better binding of spores to the biosensor's surface. Additionally, the sensors immobilized with phage under this condition were exposed to B. anthracis spores in different concentrations ranging from 5 x 10(1) to 5 x 10(8) cfu/mL in a flowing system. The results showed that the sensitivity of this ME biosensor was 202 Hz/decade.     

Ultrasensitive quartz crystal microbalance sensors for detection of M13-Phages in liquids

Quartz crystal microbalance (QCM) sensors are widely used for determining liquid properties or probing interfacial processes. For some applications the sensitivity of the QCM sensors typically used (5–20 MHz) is limited compared with other biosensor methods. In this study ultrasensitive QCM sensors with resonant frequencies from 39 to 110 MHz for measurements in the liquid phase are presented. The fundamental sensor effect of a QCM is the decrease of the resonant frequency of an oscillating quartz crystal due to the binding of mass on a coated surface during the measurement. The sensitivity of QCM sensors increases strongly with an increasing resonant frequency and, therefore, with a decreasing thickness of the sensitive area. The new kind of ultrasensitive QCM sensors used in this study is based on chemically milled shear mode quartz crystals which are etched only in the center of the blank, forming a thin quartz membrane with a thick, mechanically stable outer ring. An immunoassay using a virus specific monoclonal antibody and a M13-Phage showed an increase in the signal to noise ratio by a factor of more than 6 for 56 MHz quartz crystals compared with standard 19 MHz quartz crystals, the detection limit was improved by a factor of 200. Probing of acoustic properties of glycerol/water mixtures resulted in an increase in sensitivity, which is in very good agreement with theory. Chemically milled QCM sensors strongly improve the sensitivity in biosensing and probing of acoustic properties and, therefore, offer interesting new application fields for QCM sensors.     

The two modes of binding of Ru(phen)(2)dppz(2+) to DNA: thermodynamic evidence and kinetic studies

The binding of Ru(phen)(2)dppz(2+) (dppz=dipyrido[3,2-a:2',3'-c]phenazine) to DNA was investigated at pH 7.0 and 25 degrees C using stopped-flow and spectrophotometric methods. Equilibrium measurements show that two modes of binding, whose characteristics depend on the polymer to dye ratio (C(P)/C(D)), are operative. The binding mode occurring for values of C(P)/C(D) higher than 3 exhibits positive cooperativity, which is confirmed by kinetic experiments. The reaction parameters are K=2 x 10(3)M(-1), omega=550, n=1, k(r)=(1.9+/-0.5) x 10(7)M(-1)s(-1) and k(d)=(9.5+/-2.5)x10(3)s(-1) at I=0.012 M. The results are discussed in terms of prevailing surface interaction with DNA grooves accompanied by partial intercalation of the dppz residue. The other binding mode becomes operative for C(P)/C(D)<3 and the equilibria analysis shows this is an ordinary intercalation mode (K=1.3 x 10(6) M(-1), n=1.5 at I=0.012 M and K=2 x 10(5) M(-1), n=1.2 at I=0.21 M). Similar behaviour is displayed by double-stranded poly(A).     

Spasmolytic action of the methanol extract and isojuripidine from Solanum asterophorum Mart. (Solanaceae) leaves in guinea-pig ileum

Solanum asterophorum Mart. (Solanaceae) is a shrub popularly known as "jurubeba-defogo" in the northeast of Brazil. In the present work, the methanol extract (SA-MeOH, 3750 microg/mL) and isojuripidine (10(-7) - 3 x 10(-4) M), a steroidal alkaloid obtained from S. asterophorum Mart. leaves, inhibited phasic contractions induced by both 1 microM histamine [IC50 = (225.8 +/- 47.4), g/mL and (3.5 +/- 0.8) x 10(-5) M] or 1 microm acetylcholine [IC50 = (112.5 +/- 20.6) microg/mL and (2.3 +/- 0.4) x 10(-5) M] in guinea-pig ileum, respectively. The extract and isojuripidine also relaxed the ileum (SA-MeOH, 1-750 microg/mL, and isojuripidine, 10(-9) - 3 x 10(-4) M) pre-contracted with 1 M histamine [EC50 = (101.1 +/- 17.4) microg/mL and (1.2 +/- 0.3) x 10(-6) M] or 1 microM acetylcholine [EC50 = (136.8 +/- 21.1) microg/mL and (1.9 +/- 0.4) x 10(-6) M] or 40 mm KCl [EC50 = (149.4 +/- 19.5) microg/mL and (1.8 +/- 0.7) x 10(-6) M], respectively, in an equipotent and concentration-dependent manner. This effect is probably due to inhibition of calcium influx through voltage-operated calcium (Ca(v)) channels. To confirm this hypothesis, we evaluated their effect on cumulative CaCl2 curves in depolarizing medium nominally without Ca2+. SA-MeOH (27, 243, 500, and 750 microg/mL) and isojuripidine (3 x 10(-8), 10(-6), 3 x 10(-5), and 3 x 10(-4) M) inhibited the contractions induced by CaCl2, in a concentration-dependent manner. The concentration-response curves to CaCl2, in the presence of SA-MeOH and isojuripidine, were shifted downward in relation to a control curve in a non-parallel manner resulting in reduction of the maximum effect [E(max) = (71.2 +/- 9.2); (57.4 +/- 9.2); (43.8 +/- 3.4); (41.5 +/- 2.4) and (90.6 +/- 4.8); (74.7 +/- 8.7); (66.4 +/- 3.9); (31.3 +/- 4.1)%, respectively]. SA-MeOH and isojuripidine present spasmolytic action in guinea-pig ileum due to a partially blockade of calcium influx through Ca(v) channels.     

蝎毒耐热蛋白对大鼠急性分离海马神经元兴奋性的影响

应用全细胞膜片钳记录技术在电流钳模式下观察经持续高温等特殊处理后分离纯化的30～50 kDa蝎毒耐热蛋白(scorpion venom heat resistant protein,SVHRP)(国家发明专利,专利号ZL01 106166.92)对急性分离大鼠海马神经元兴奋性的影响.结果发现SVHRP可致海马神经元兴奋性降低.神经元经1×10-2 μg/mL SVHRP处理后动作电位发放模式改变,发放频率减少.在52个受检细胞中,有45个细胞产生位相放电(占86.54%);7个细胞产生重复放电(占13.46%).在产生位相放电的45个细胞中,有8个细胞在SVHRP处理后仍可以诱发出位相放电(占17.78%);37个细胞在SVHRP处理后无法诱导出位相放电(占82.22%),SVHRP处理后动作电位的产生与处理前相比,有显著差异(P＜0.01,n=45);在产生重复放电的7个细胞中,在1×10-2μg/mL SVHRP作用后均不能再次诱发出重复放电,而是产生一个动作电位或不再产生动作电位,药物处理前产生的动作电位个数为14.57±1.00,SVHRP处理后产生动作电位的个数为0.57±0.20,二者之间有显著性差异(P＜0.01,n=7).1×10-4 μg/mLSVHRP处理后,诱发动作电位产生的基强度由(75.10±8.99)pA增加到(119.85±12.73)pA(P＜0.01,n=8);阈电位由(-41.17±2.15)mV升至(-32.40±1.48)mV(P＜0.01,n=8);动作电位峰值由(68.49±2.33)mV下降至(54.71±0.81)mV(P＜0.01,n=8).由于神经元超兴奋性被认为是癫痫发作的基本机制之一,因此上述结果表明SVHRP有可能通过降低海马神经元兴奋性发挥其抗癫痫作用,这为蝎毒药物的进一步开发提供理论依据.     

Hepatocyte viability and protein expression within hydrogel microstructures

Poly(ethylene) glycol (PEG) hydrogels have been successfully used to entrap mammalian cells for potential high throughput drug screening and biosensing applications. To determine the influence of PEG composition on the production of cellular protein, mammalian hepatocytes were maintained in PEG hydrogels for 7 days. Total cell viability, total protein production, and the production of two specific proteins, albumin and fibronectin, were monitored. Studies revealed that while PEG composition has no effect on cell viability, increasing amounts of PEG in the hydrogel decrease the amount of protein production by the cells after 7 days from 1.0 x 10(5) +/- 1.7 x 10(4) to 5.2 x 10(3) +/- 1.3 x 10(3) g accumulated protein/mL/million cells. Additionally, cells entrapped in PEG hydrogels produce greater amounts of protein than traditional monolayer culture (1.5 x 10(3) +/- 1.9 x 10(2) g accumulated protein/mL/million cells after 7 days). The addition of the synthetic peptide RGD to 10% PEG hydrogels altered the production of the proteins albumin and fibronectin. Hydrogels with the RGD sequence produced 287 +/- 27 ng/mL/million cells albumin after 7 days, an order of magnitude greater than monolayer cultures, whereas cells in hydrogels without the RGD sequence produced undetectable levels of albumin. Conversely, cells entrapped in 10% PEG hydrogels without the RGD sequence produced 1014 +/- 328 ng/mL/million cells fibronectin after 7 days, whereas 10% PEG hydrogels with the RGD sequence produced 200 +/- 58 ng/mL/million cells fibronectin after 7 days.     

IGF-I, IGFBP-2 and -3 but not GH concentrations are different in normal and poor growing piglets.

In this study we investigated the somatotropic axis in piglets with evident growth delay. Female Suffolk crossbred piglets (30 days old; N = 12) were divided into normal weight (10 +/- 0.9 kg) and poor growing subjects (7 +/- 0.5 kg) and bled for growth hormone (GH), Insulin-like growth factor-I (IGF-I), Insulin-like growth factor binding protein 2 and 3 (IGFBP-2 and -3) determination. Basal and induced-GH levels were not different in the groups. Plasma IGF-I concentrations were significantly different (p < 0.001): 101.8 +/- 9.8 ng x mL(-1) (normal weight group) and 39.5 +/- 4.0 ng x mL(-1) (poor growing group). IGFBP-2 and -3 concentrations were significantly (p < 0.001) lower in poor growing than in normal piglets. Piglet weight was positively correlated (r = 0.98, p < 0.001) with IGF-I and IGFBP-2 or -3 concentrations. Our data indicate that growth rate was not correlated to basal or secretagogue-induced GH secretion.     

Measurement of mass transport and reaction parameters in bulk solution using photobleaching. Reaction limited binding regime.

Fluorescence recovery after photobleaching (FRAP) has been used previously to investigate the kinetics of binding to biological surfaces. The present study adapts and further develops this technique for the quantification of mass transport and reaction parameters in bulk media. The technique's ability to obtain the bulk diffusion coefficient, concentration of binding sites, and equilibrium binding constant for ligand/receptor interactions in the reaction limited binding regime is assessed using the B72.3/TAG-72 monoclonal antibody/tumor associated antigen interaction as a model in vitro system. Measurements were independently verified using fluorometry. The bulk diffusion coefficient, concentration of binding sites and equilibrium binding constant for the system investigated were 6.1 +/- 1.1 x 10(-7) cm2/s, 4.4 +/- 0.6 x 10(-7) M, and 2.5 +/- 1.6 x 10(7) M-1, respectively. Model robustness and the applicability of the technique for in vivo quantification of mass transport and reaction parameters are addressed. With a suitable animal model, it is believed that this technique is capable of quantifying mass transport and reaction parameters in vivo.     

Divalent ion-binding properties of the two avian beta-parvalbumins

Birds express three parvalbumins, one alpha isoform and two beta isoforms. The latter are known as avian thymic hormone (ATH) and avian parvalbumin 3. Although both were discovered in thymus tissue, and presumably function in T-cell maturation, they have been detected in other tissue settings. We have conducted detailed Ca2+- and Mg2+-binding studies on recombinant ATH and the C72S variant of CPV3, employing global analysis of isothermal titration calorimetry data. In Hepes-buffered saline, ATH binds Ca2+ with apparent microscopic binding constants of 2.4 +/- 0.2 x 10(8) and 1.0 +/- 0.1 x 10(8) M(-1). The corresponding values for CPV3-C72S are substantially lower, 4.5 +/- 0.5 x 10(7) and 2.4 +/- 0.2 x 10(7) M(-1), a 1.9-kcal/mol difference in binding free energy. Thus, the beta-parvalbumin lineage displays a spectrum of Ca2+-binding affinity, with ATH and the mammalian beta isoform at the high- and low-affinity extremes and CPV3 in the middle. Interestingly, despite its decreased Ca2+ affinity, CPV3-C72S exhibits increased affinity for Mg2+, relative to ATH. Whereas the latter displays Mg2+-binding constants of 2.2 +/- 0.2 x 10(4) and 1.2 +/- 0.1 x 10(4) M(-1), CPV3-C72S yields values of 5.0 +/- 0.8 x 10(4) and 2.1 +/- 0.3 x 10(4) M(-1).     

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.     

Immunoassay of prostate-specific antigen (PSA) using resonant frequency shift of piezoelectric nanomechanical microcantilever

We designed and fabricated the nanomechanical Pb(Zr0.52Ti0.48)O3 (PZT) cantilever; we demonstrated a novel electrical measurement, under a controlled ambient temperature and humidity, for label-free detection of a prostate-specific antigen (PSA); and we achieved a detection sensitivity as low as 10 pg/ml. For the fabrication of our nanomechanical PZT cantilevers, we used composite layers of Ta/Pt/PZT/Pt/SiO2 on a SiN(x) supporting layer for electrical self-sensing without external oscillators. This method allows PSA proteins to be detected via a simple electrical measurement of the resonant frequency change generated by the molecular interaction of the antigen (Ag) and the antibody (Ab). The resonant frequency shifted due to the specific binding of the PSA Ag to its Ab which is immobilized via calixcrown self-assembled monolayers on an Au surface deposited on a nanomechanical PZT cantilever. We determined the resonant frequency shift as the value of -172 Hz and -273 Hz, when the concentration of PSA Ag was 1 ng/ml, with the cantilever dimensions of 100 microm x 300 microm and 50 microm x 150 microm, respectively. Theoretical and experimental analysis suggests that the minimum detectable sensitivity for a resonant frequency shift due to a PSA Ag-Ab interaction depends on the dimensions of the nanomechanical PZT cantilever. These results also demonstrate that the experimentally measured resonant frequency shift is larger than that calculated theoretically due to the compressive stress of the PSA Ag-Ab interaction.