Impedimetric immunosensor doped with reduced graphene sheets fabricated by controllable electrodeposition for the non-labelled detection of bacteria

A facile, sensitive and reliable impedimetric immunosensor doped with reduced graphene sheets (RGSs) and combined with a controllable electrodeposition technique was developed for the selective detection of marine pathogenic sulphate-reducing bacteria (SRB). The morphology of RGSs and the electrochemical properties of RGSs-doped chitosan (CS) nanocomposite film were investigated by atomic force microscopy, Fourier transform infrared spectroscopy, and cyclic voltammetry (CV). Electrochemical impedance spectroscopy and CV were used to verify the stepwise assembly of the sensor system. Faradic impedance spectroscopy for charge transfer for the redox probe Fe(CN)(6)(3-/4-) was done to determine SRB concentrations. The diameter of the Nyquist diagram that is equal to the charge-transfer resistance (R(ct)) increased with increasing SRB concentration. A linear relationship between R(ct) and SRB concentration was obtained in the SRB concentration range of 1.8×10(1) to 1.8×10(7) cfu/ml. The impedimetric biosensor gave a distinct response to SRB, but had no obvious response to Vibrio angillarum. It showed a high selectivity for the detection of the pathogen. Based on a combination of the biocompatibility of CS and good electrical conductivity of RGSs, a nanocomposite film with novel architecture was used to immobilize biological and chemical targets and to develop a new type of biosensor.     

Direct immobilisation of antibodies on a bioinspired architecture as a sensing platform

A sensitive and selective immunosensor for the nonlabeled detection of sulfate-reducing bacteria (SRB) is constructed using a self-polymerised polydopamine film as the immobilisation platform. Self-polymerisation of dopamine is used as a powerful approach for applying multifunctional coatings onto the surface of a gold electrode. The polydopamine film is used not only as the immobilisation platform, but also as a cross-linker reagent for the immobilisation of the anti-SRB antibody. The polydopamine film is loaded with a high density of anti-SRB antibodies linked to the substrate to obtain high response signals. The formation and fabrication of the biosensor and the quantification of antibody anchoring are monitored, and SRB detection is performed by either quartz crystal microbalance (QCM) or electrochemical impedance spectroscopy (EIS). After modeling the impedance Nyquist plots of the SRB/anti-SRB/polydopamine/gold electrode for increasing concentrations of SRB, the electron transfer resistance (R(ct)) is used as a measure of immunocomplex binding. The R(ct) is correlated with the concentration of bacterial cells in the range of 1.8×10(2) to 1.8×10(6) CFU mL(-1); the detection limit is 50 CFU mL(-1). This work demonstrates a new immobilisation platform for the development of a sensitive and label-less impedimetric and piezoelectric immunosensor. This immunosensor may be broadly applied in clinical diagnoses and the monitoring of water environmental pollution. The method proposed is distinct in its ease of application, use of a simple protocol, and mild reaction conditions. These allow it to be applied to a wide variety of materials.     

Electrochemical imprinted sensor for determination of oleanic acid based on poly (sodium 4-styrenesulfonate-co-acrylic acid)-grafted multi-walled carbon nanotubes-chitosan and cobalt hexacyanoferrate nanoparticles

A novel sensitive and selective imprinted electrochemical sensor for the determination of oleanic acid was constructed on a carbon electrode by stepwise modification of functional multi-walled carbon nanotubes, cobalt hexacyanoferrate nanoparticles and a thin imprinted sol-gel film. The fabrication of a homogeneous porous poly (sodium 4-styrenesulfonate-co-acrylic acid)-grafted multi-walled carbon nanotubes/SiO(2)-chitosan nanocomposite film was conducted by controllable electrodeposition technology. The surface morphologies of the modified electrodes were characterized by scanning electron microscope. The performance of the imprinted sensor was investigated by cyclic voltammetry, square wave voltammetry and electrochemical impedance spectroscopy in detail. The imprinted sensor displayed high sensitivity and selectivity towards oleanic acid. A linear relationship between the sensor response signal and the logarithm of oleanic acid concentrations ranging from 1.0×10(-8) to 1.0×10(-3) mol L(-1) was obtained with a detection limit of 2.0×10(-9) mol L(-1). It was applied to the determination of oleanic acid in real capsule samples successfully.     

Stereoselective histidine sensor based on molecularly imprinted sol-gel films

A piezoelectric sensor coated with a thin molecularly imprinted sol-gel film has been developed for the determination of L-histidine in the liquid phase. Without preprotection, L-histidine was imprinted directly into silica sol-gel films that consisted of a hybrid mixture of functionalized organosilicon precursors (phenyltrimethoxysilane and methyltrimethoxysolane). The viscoelasticity of the film in the air and in buffer solution has been studied by the piezoelectric quartz crystal impedance technique. The binding of L-histidine to the imprinted film in the liquid phase was investigated by the piezoelectric microgravimetry and electrochemical impedance technique. Scatchard analysis showed that the maximum binding site (Qmax) of the L-histidine imprinted sol-gel film is about 23.7 micromol/g. A linear range from 5.0x10(-8) to 1.0x10(-4) M for a detection of L-histidine has been observed with a detection limit of 2.5x10(-8) M for S/N=3. The proposed imprinted sol-gel sensor exhibits good stability, high specificity, and excellent stereoselectivity.     

Chitosan/polyaniline hybrid conducting biopolymer base impedimetric immunosensor to detect Ochratoxin-A

Chitosan (CS)-polyaniline (PANI) hybrid conducting biopolymer film was obtained on indium-tin-oxide (ITO) electrode using electrochemical polymerization process. Fourier transform infrared (FT-IR) spectra of PANI-CS had showed covalent and hydrogen binding between PANI and CS molecules. Electrochemical impedance spectroscopy (EIS) measurements had showed low charge transfer resistance (R(CT)) of PANI-CS and PANI. Successive rabbit antibody (IgGs) immobilization on PANI-CS, CS and PANI matrixes surface were confirmed with FT-IR and EIS measurements. Ochratoxin-A (OTA) interaction with IgGs had increased R(CT) values and showed linear response up to 10 ng/mL OTA concentration in electrolyte. Relative change in R(CT) was higher in PANI-CS due to higher proportion of carboxylic and hydroxyl functionalities at PANI-CS matrix surfaces. The absolute sensitivity of PANI, CS, and PANI-CS were 16+/-6, 22+/-9 and 53+/-8 Omega mL/ng, respectively derived from slope of linear response up to 10 ng/mL with 1 ng/mL minimum detection limit.     

Nanostructured conducting molecularly imprinted polymer for selective uptake/release of naproxen by the electrochemically controlled sorbent

A conducting molecularly imprinted polymer (CMIP) film, based on polypyrrole, was electrosynthesized for selective uptake/release and determination of naproxen. The film was prepared by incorporation of a template anion (naproxen) during the electropolymerization of pyrrole into a platinum electrode using the cyclic voltammetry method. Overoxidized polypyrrole films with cavities complementary to the template were used as a potential-induced selective recognition element in the solid-phase sorbent. Various important fabricating factors, which control the performance of the CMIP film, were investigated using fluorescence spectroscopy. The measured fluorescence intensities of released solutions were related to the concentrations of naproxen taken up into the films. Several key parameters such as applied potential and time for uptake and release were varied to achieve the optimal sorption procedure. The film template with naproxen exhibited excellent selectivity over some interference. The calibration graphs were linear in the ranges of 5×10(-8) to 3×10(-7)molml(-1) and 7×10(-6) to 8×10(-4)molml(-1), and the limit of detection was 1×10(-8)molml(-1). The CMIP films, as the electrochemically controlled solid-phase sorbent, were applied for the selective cleanup and quantification of trace amounts of naproxen from physiological samples. Scanning electron microscopy confirmed the nanostructure morphology of the films.     

Ultra trace analysis of small molecule by label-free impedimetric immunosensor using multilayer modified electrode

A multilayer electrode modified with a self-assembled thiourea monolayer (SATUM) followed by gold nanoparticles (AuNPs), mercaptosuccinic acid (MSA) and antibody was investigated for the detection of ultra trace amount of a small molecule (chloramphenicol) in an impedimetric system. The formation of the antibody-antigen complex at the electrode surface caused the impedance to increase. Under optimum conditions three modified electrodes were compared the SATUM/AuNPs/MSA electrode provided a wide linear range (0.50-10) × 10?1? M, and a very low determination limit of 1.0 × 10?1? M. This determination limit was much lower than the SATUM/AuNPs electrode, 1.0 × 10?1? M, and SATUM electrode, 4.7 × 10?1? M. The modified electrode provided good selectivity for chloramphenicol detection and can be reused up to 45 times with a relative standard deviation of lower than 4%. When applied to determine chloramphenicol in shrimp samples, the results agreed well with those obtained by the high-performance liquid chromatography coupled with a photo diode array detector (P > 0.05). The developed system can be applied to detect other small molecules using appropriate affinity binding pairs.     

长双歧杆菌DD98冻干保护剂优化及菌粉保存稳定性研究

以长双歧杆菌DD98为研究对象,通过对冻干保护剂配方的优化,冻干菌粉的存活率提高到90%以上。通过进一步稳定性研究,采用保护剂优化配方制备的冻干菌粉在4℃保存24个月后,活菌数仍在1.0×10^10 CFU/g以上,在25℃条件下可以保存12个月,双歧杆菌的存活率在1.0×10^6CFU/g以上,符合FAO/WHO建议食品益生菌活菌数应在1.0×10^6 CFU/g^1.0×10^7CFU/g的标准。     

Detection of viable Salmonella using microelectrode-based capacitance measurement coupled with immunomagnetic separation

In this study, we demonstrated the use of a general medium--brain heart infusion (BHI) broth that is not specifically formulated for impedance measurement, to achieve detectable impedance signals by using an interdigitated microelectrode (IME) with capacitance measurement at low frequencies. Anti-Salmonella antibody coated immunomagnetic beads were used to separate S. typhimurium from samples to provide the selectivity to this method. From analysis based on the equivalent circuit of the IME system, we found that the impedance change in BHI broth resulting from the growth of Salmonella was indeed the change in the double layer capacitance and could be monitored at 10 Hz using the IME. The results indicated that medium modification to improve impedance signal is not necessary with this IME system. However, effective immunological separation for the target organism is required for the selectivity when non-selective media are used. This finding provides a more flexible option of medium in impedance methods, which may provide opportunities to test those species of bacteria that have no suitable conductance growth medium. The detection time, t(d), was obtained from the impedance growth curve (impedance against bacterial growth time) at 10 Hz at the point where the impedance started to change. A linear relationship between the detection time and the logarithmic value of the initial cell number (N) was found in the Salmonella cell number ranging from 10(1) to 10(6) cfu/ml. The regression equation was t(d) = -1.22Log N + 8.90, with R2 = 0.95. The detection times for the initial cell number of 10(1) CFU/ml and 10(6) CFU/ml are 8 h and 1.5 h, respectively. This method is more sensitive than impedance methods using conventional electrodes.     

Quantification of toxic and inhibitory impact of copper and zinc on mixed cultures of sulfate-reducing bacteria

The adverse effects of copper and zinc on an acetate-utilizing mixed cultures of sulfate-reducing bacteria (SRB) at concentrations below the toxic concentration (minimum metal concentration at which no sulfate reduction is observed) are reported in this paper. Mathematical models were developed to incorporate the toxic and inhibitory effects (defined as the reduction in bacterial population upon exposure to the metal and the decrease in the metabolic rate of sulfate reduction by the SRB, respectively) into the sulfate-reduction biokinetics. The characteristic toxicity and inhibition constants were obtained from the measurements of bacterial populations and dissolved metal concentrations in serum bottle studies conducted at 35 degrees C and pH 6.6. Both copper and zinc had toxic and inhibitory effects on SRB. The toxicity constants for copper and zinc were 10.6 and 2.9 mM(-1), respectively, indicating that exposure to copper resulted in a higher mortality of SRB than did exposure to zinc. The values of the inhibition constants were found to be 17.9 +/- 2.5 and 25.2 +/- 1.0 mM(-1) for copper and zinc, respectively. This implies that dissolved zinc was slightly more inhibitory to SRB than copper. The models presented in the paper can be used to predict the response of a sulfate-reduction bioreactor to heavy metals during acid mine drainage treatment.     

DNA electrochemical biosensor based on thionine-graphene nanocomposite

A novel protocol for development of DNA electrochemical biosensor based on thionine-graphene nanocomposite modified gold electrode was presented. The thionine-graphene nanocomposite layer with highly conductive property was characterized by scanning electron microscopy, transmission electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. An amino-substituted oligonucleotide probe was covalently grafted onto the surface of the thionine-graphene nanocomposite by the cross-linker glutaraldehyde. The hybridization reaction on the modified electrode was monitored by differential pulse voltammetry analysis using an electroactive intercalator daunomycin as the indicator. Under optimum conditions, the proposed biosensor exhibited high sensitivity and low detection limit for detecting complementary oligonucleotide. The complementary oligonucleotide could be quantified in a wide range of 1.0 × 10(-12) to 1.0 × 10(-7)M with a good linearity (R(2)=0.9976) and a low detection limit of 1.26 × 10(-13)M (S/N=3). In addition, the biosensor was highly selective to discriminate one-base or two-base mismatched sequences.     

Culture-dependent comparison of microbial diversity in deep granitic groundwater from two sites considered for a Swedish final repository of spent nuclear fuel

Site selection for a spent nuclear fuel (SNF) repository required analysis of microbial abundance and diversity at two Swedish sites, Forsmark and Laxemar-Simpevarp. Information about sulphate-reducing bacteria (SRB) was required, as sulphide could corrode copper SNF canisters. Total number of cells (TNC) and ATP were analysed, and plate counts and most probable number (MPN) analyses were conducted using eight media based on different electron donors and acceptors for specific microorganism physiological groups. Groundwater chemical composition and E(h) were analysed; sampling depths were 112-978 m below sea level. TNC was 5.5 × 10(3) to 4.7 × 10(5) cells mL(-1), correlating with ATP concentrations. Culturability in TNC percentage was 0.01-35.9, averaging 5.12. Culturable numbers varied greatly between sample positions and uncorrelated with depth. SRB were found in 29 samples and were below detection in three; the MPN of SRB correlated negatively with E(h), as did the MPN of acetogens. Data indicated that microbial sulphate reduction was ongoing in many sampled aquifers; published stable isotope data and modelling results supported this observation. The sites did not differ significantly, but the large data range suggested that analysis of more samples would enable detailed evaluation of microbial processes and their relationship with geochemical information.     

The use of alkaline phosphatase-labelled oligonucleotide probes as culture confirmation reagents for the identification of commercially important bacteria

A range of rRNA-targeted alkaline phosphatase-labelled oligonucleotide probes was tested for use as culture confirmation reagents for the rapid identification of micro-organisms. The probes were specific to clinically important bacteria ( Helicobacter pylori and Mycobacterium tuberculosis ), fish and shellfish pathogens ( Renibacterium salmoninarum and Vibrio vulnificus ), food spoilage bacteria ( Listeria spp. and L. monocytogenes ), for bacteria of biotechnological importance ( Streptomyces spp.) and for bacteria associated with the oil industry (Sulphate-reducing bacteria, SRB). A universal bacterial probe and a eukaryotic probe were included in the study as positive and negative controls, respectively. A total of 93 bacterial strains was screened. With the exception of a large number of cross-reactions of the SRB probe (specificity value of 29·4%) and a single cross-reaction of the R. salmoninarum probe (specificity value of 97·7%), dot blot analysis indicated that each probe hybridized 100% specifically to the organisms tested. A simple culture confirmation method was then developed using these probes to enable the identification of bacterial colonies using a simple hybridization procedure.     

Ellipsometric Measurement of Bacterial Films at Metal-Electrolyte Interfaces

Ellipsometric measurements were used to monitor the formation of a bacterial cell film on polarized metal surfaces (Al-brass and Ti). Under cathodic polarization bacterial attachment was measured from changes in the ellipsometric angles. These were fitted to an effective medium model for a nonabsorbing bacterial film with an effective refractive index (n_f) of 1.38 and a thickness (d_f) of 160 ± 10 nm. From the optical measurements a surface coverage of 17% was estimated, in agreement with direct microscopic observations. The influence of bacteria on the formation of oxide films was monitored by ellipsometry following the film growth in situ. A strong inhibition of metal oxide film formation was observed, which was assigned to the decrease in oxygen concentration due to the presence of bacteria. It is shown that the irreversible adhesion of bacteria to the surface can be monitored ellipsometrically. Electrophoretic mobility is proposed as one of the factors determining bacterial attachment. The high sensitivity of ellipsometry and its usefulness for the determination of growth of interfacial bacterial films is demonstrated.     

Interdigitated microelectrode (IME) impedance sensor for the detection of viable Salmonella typhimurium

Interdigitated microelectrodes (IMEs) were used as impedance sensors for rapid detection of viable Salmonella typhimurium in a selective medium and milk samples. The impedance growth curves, impedance against bacterial growth time, were recorded at four frequencies (10Hz, 100Hz, 1kHz, and 10kHz) during the growth of S. typhimurium. The impedance did not change until the cell number reached 10(5)-10(6) CFUml(-1). The greatest change in impedance was observed at 10Hz. To better understand the mechanism of the IME impedance sensor, an equivalent electrical circuit, consisting of double layer capacitors, a dielectric capacitor, and a medium resistor, was introduced and used for interpreting the change in impedance during bacterial growth. Bacterial attachment to the electrode surface was observed with scanning electron microscopy, and it had effect on the impedance measurement. The detection time, t(D), defined as the time for the impedance to start change, was obtained from the impedance growth curve at 10Hz and had a linear relationship with the logarithmic value of the initial cell number of S. typhimurium in the medium and milk samples. The regression equations for the cell numbers between 4.8 and 5.4 x 10(5) CFUml(-1) were t(D) = -1.38 log N + 10.18 with R(2) = 0.99 in the pure medium and t(D) = -1.54 log N + 11.33 with R(2) = 0.98 in milk samples, respectively. The detection times for 4.8 and 5.4 x 10(5) CFUml(-1) initial cell numbers were 9.3 and 2.2 h, respectively, and the detection limit could be as low as 1 cell in a sample.     

A selective novel non-enzyme glucose amperometric biosensor based on lectin-sugar binding on thionine modified electrode

A novel non-enzyme glucose amperometric biosensor was fabricated based on biospecific binding affinity of concanavalin A (Con A) for D-glucose on thionine (TH) modified electrode. TH can be covalently immobilized on potentiostatically activated glassy carbon electrode through Schiff-base reaction. Subsequently, the surface-adherent polydopamine film formed by self-polymerization of dopamine attached to TH and afforded binding sites for the subsequent immobilization of Con A molecules via Michael addition and/or Schiff-base reaction with high stability. Thus, a sensing platform for specific detection towards D-glucose was established. The binding of Con A towards D-glucose can be monitored through the decrease of the electrode response of the TH moiety. Due to the high affinity of Con A for D-glucose and high stability of the resulting sensing platform, the fabricated biosensor exhibited high selectivity, good sensitivity, and wide linear range from 1.0×10(-6) to 1.0×10(-4) M with a low detection limit of 7.5×10(-7) M towards D-glucose.     

Detection of viable Salmonella typhimurium by impedance measurement of electrode capacitance and medium resistance

Three-electrode electrochemical impedance technique was investigated for detection of Salmonella typhimurium by monitoring the growth of bacteria in selenite cystine (SC) broth supplemented with trimethylamine oxide hydrochloride (TMAO.HCl) and mannitol (M). The change in the system impedance during the growth of bacteria was studied using frequency spectral scanning. It was found that the impedance at low frequencies (<10 kHz) mainly came from the double-charged layer capacitance, reflecting the changes at the electrode interface and the adsorption on the electrode surface. While at high frequencies (>10 kHz), the system impedance mainly depended on the medium resistance. The adsorption of bacteria on the electrode surface was detected by measuring low frequency impedance, and verified with Faradic impedance spectroscopy. Enumeration of S. typhimurium using a low frequency (1 Hz) capacitance measurement and a high frequency (1 MHz) resistance measurement were compared. The detection times were determined for quantitative analysis based on the growth curves of bacteria referring to either the medium resistance or electrode capacitance. The regression equations for the detection times (t(d), h) and the initial cell number (N, cells.ml(-1)) were t(d)=-1.24logN+13.4 with R(2)=0.98 and t(d)=-1.40logN+14.46 with R(2)=0.97 for the medium resistance and electrode capacitance methods, respectively.     

Three-step electrodeposition synthesis of self-doped polyaniline nanofiber-supported flower-like Au microspheres for high-performance biosensing of DNA hybridization recognition

A three-step electrodeposition method has been successfully adopted to fabricate morphology-controlled novel Au microspheres on self-doped polyaniline nanofibers (nanoSPAN) modified glassy carbon electrode. The deposition conditions, such as HAuCl(4) concentration and deposition step, have significant influences on the morphologies and electrochemical properties of the resulted Au microspheres. Well hierarchical and homogeneously dispersed flower-like Au microspheres (HHFAu) were obtained under optimal conditions by the three-step electrodeposition strategy in 5.0mM HAuCl(4) solution. HHFAu possess large surface area, excellent electron transfer ability and good biocompatibility. The DNA probe could be effectively attached to HHFAu and thus a high-performance DNA biosensor was constructed by using electrochemical impedance spectroscopy as detection method. A gene fragment of the cauliflower mosaic virus 35S gene, which is related to one of the screening genes for the transgenically modified plants, has been satisfactorily detected. The linear range was from 1.0 × 10(-13)M to 1.0 × 10(-6)M and the detection limit was 1.9 × 10(-14)M. This HHFAu/nanoSPAN-based impedance biosensing platform holds great promise for the detection of other biological and chemical molecules.     

Colorimetric/fluorescent bacterial sensing by agarose-embedded lipid/polydiacetylene films

Aim: Development of a new chromatic (colorimetric/fluorescence) bacterial sensor, for rapid, sensitive and versatile detection of bacterial proliferation. Methods and Results: We constructed agarose‐embedded chromatic films which produce dramatic colour changes and fluorescence transformations in response to bacterial growth. The sensing constructs comprise glass‐supported Langmuir–Schaeffer phospholipid/polydiacetylene films that undergo both blue‐red transformations and induction of intense fluorescence following interactions with bacterially secreted amphiphilic compounds that diffuse through the agarose. The agarose matrix coating the sensor film further contains growth nutrients, facilitating signal amplification through promotion of bacterial culture proliferation. The agarose layer also constitutes an effective barrier for reducing background signals not associated with the bacteria. We demonstrate the applications of the new sensor for the detection of Gram‐negative and Gram‐positive bacteria, and for screening specimens of physiological fluids (blood and urine) and foods (meat) for bacterial contaminations. Conclusions: The experiments demonstrate that the new agarose‐embedded film constructs are capable of bacterial detection through visible colour transitions and fluorescence emission recorded in conventional apparatuses. Significance and Impact of the Study: This work demonstrated a new simple chromatic platform for bacterial detection, based on the generation of easily recorded colour and fluorescence changes. The new bacterial detection scheme is highly generic and could be employed for varied practical uses, in which, rapid reporting on bacterial presence is required.     

A high-sensitive and fast-fabricated glucose biosensor based on Prussian blue/topological insulator Bi(2)Se(3) hybrid film

A novel and fast-fabricated Prussian blue (PB)/topological insulator Bi(2)Se(3) hybrid film has been prepared by coelectrodeposition technique. Taking advantages of topological insulator in possessing exotic metallic surface states with bulk insulating gap, Prussian blue nanoparticles in the hybrid film have smaller size as well as more compact structure, showing excellent pH stability even in the alkalescent solution of pH 8.0. Based on the Laviron theory, the electron transfer rate constant of PB/Bi(2)Se(3) hybrid film modified electrode was calculated to be 4.05±0.49s(-1), a relatively big value which may be in favor of establishing a high-sensitive biosensor. An amperometric glucose biosensor was then fabricated by immobilizing glucose oxidase (GOD) on the hybrid film. Under the optimal conditions, a wide linear range extending over 3 orders of magnitude of glucose concentrations (1.0×10(-5)-1.1×10(-2)M) was obtained with a high sensitivity of 24.55μAmM(-1) cm(-2). The detection limit was estimated for 3.8μM defined from a signal/noise of 3. Furthermore, the resulting biosensor was applied to detect the blood sugar in human serum samples without any pretreatment, and the results were comparatively in agreement with the clinical assay.