Dual gold nanoparticle lateflow immunoassay for sensitive detection of Escherichia coli O157:H7

Two patterns of signal amplification lateral flow immunoassay (LFIA), which used anti-mouse secondary antibody-linked gold nanoparticle (AuNP) for dual AuNP-LFIA were developed. Escherichia coli O157:H7 was selected as the model analyte. In the signal amplification direct LFIA method, anti-mouse secondary antibody-linked AuNP (anti-mouse-Ab-AuNP) was mixed with sample solution in an ELISA well, after which it was added to LFIA, which already contained anti-E. coli O157:H7 monoclonal antibody-AuNP (anti-E. coli O157:H7-mAb-AuNP) dispersed in the conjugate pad. Polyclonal antibody was the test line, and anti-mouse secondary antibody was the control line in nitrocellulose (NC) membrane. In the signal amplification indirect LFIA method, anti-mouse-Ab-AuNP was mixed with sample solution and anti-E. coli O157:H7-mAb-AuNP complex in ELISA well, creating a dual AuNP complex. This complex was added to LFIA, which had a polyclonal antibody as the test line and secondary antibody as the control line in NC membrane. The detection sensitivity of both LFIAs improved 100-fold and reached 1.14 × 10³ CFU mL⁻¹. The 28 nm and 45 nm AuNPs were demonstrated to be the optimal dual AuNP pairs. Signal amplification LFIA was perfectly applied to the detection of milk samples with E. coli O157:H7 via naked eye observation.     

An easily-automated assay for the physiological state quantification of Pseudomonas sp. M18

In order to foreknow poorly performing cultures before wasting energy to scale them to large cultures, industrial microbial fermentation can greatly benefit from knowledge of the physiological state of cells. The method currently proposed is an easily automated physiological state determination method. We have designed one universal rRNA-specific probe for bacteria and developed novel signal probe hybridization (SPH) assay featuring no RNA extraction and no PCR amplification steps necessary to quantify the physiological state of microbial cells. The microbial cell was lysed with sonication and SDS. Signal probes were applied to hybridize and protect the rRNA target. S1 nuclease was then applied to remove the excessive signal probes, the single-stranded RNA and the mismatch RNA/DNA hybrids. The remaining signal probe was captured with a corresponding capture probe immobilized on a microplate and quantified with a horseradish peroxidase-conjugated color reaction. We then systemically optimized our assay. Results showed that the cell limit of detection (LOD) and the cell limit of quantification (LOQ) were 2.64 × 10⁴ cells and 9.86 × 10⁴ cells per well of microplate, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of signal probe were 49.0 fM and 344.0 fM respectively. Using this technique, we quantified the 16S rRNA levels during the fermentation process of Pseudomonas sp. M18. Our results indicate that the 16S rRNA levels can directly inform us about the physiological state of microbial cells. This technique has great potential for application to the microbial fermentation industry.     

Application of surface plasmon resonance spectroscopy for adsorption studies of different types of components on poly(dimethylsiloxane)

Surface plasmon resonance (SPR) spectroscopy is utilized to study in real-time and, by label-free means, the reversible and quasi-irreversible adsorption of small ionic or neutral molecules, pharmaceuticals, and proteins on poly(dimethylsiloxane) (PDMS) surfaces. The SPR sensor is covered with 0.2% (w/v) PDMS in octane. During the timescale of a typical lab-on-a-chip analysis or an electrophoretic separation, it was found that small neutral components containing a hydrophobic part do not adsorb or absorb onto PDMS, while larger, water-soluble polymer-like materials like proteins generally irreversibly adsorb to PDMS. The technique can be used to monitor the kinetics of adsorption and desorption of the molecules. For the non-specific adsorption of teicoplanin to PDMS, a Langmuir-like adsorption isotherm was obtained (K_d = 32 ± 2 μmol L⁻¹).     

Visual and surface plasmon resonance sensor for zirconium based on zirconium-induced aggregation of adenosine triphosphate-stabilized gold nanoparticles

Owing to its high affinity with phosphate, Zr(IV) can induce the aggregation of adenosine 5′-triphosphate (ATP)-stabilized AuNPs, leading to the change of surface plasmon resonance (SPR) absorption spectra and color of ATP-stabilized AuNP solutions. Based on these phenomena, visual and SPR sensors for Zr(IV) have been developed for the first time. The A_660 nm/A_518 nm values of ATP-stabilized AuNPs in SPR absorption spectra increase linearly with the concentrations of Zr(IV) from 0.5 μM to 100 μM (r = 0.9971) with a detection limit of 95 nM. A visual Zr(IV) detection is achieved with a detection limit of 30 μM. The sensor shows excellent selectivity against other metal ions, such as Cu²⁺, Fe³⁺, Cd²⁺, and Pb²⁺. The recoveries for the detection of 5 μM, 10 μM, 25 μM and 75 μM Zr(IV) in lake water samples are 96.0%, 97.0%, 95.6% and 102.4%, respectively. The recoveries of the proposed SPR method are comparable with those of ICP-OES method.     

A 3,3′-dichlorobenzidine-imprinted polymer gel surface plasmon resonance sensor based on template-responsive shrinkage

Molecularly imprinted polymer gel film on the gold substrate of a chip was prepared with minute amount of cross-linker for the fabrication of a surface plasmon resonance (SPR) sensor sensitive to 3,3′-dichlorobenzidine. The molecularly imprinted gel film was anchored on a gold chip by a surface-bound photo-radical initiator. The sensing of 3,3′-dichlorobenzidine is based on responsive shrinkage of the imprinted polymer gel film that is triggered by target binding. This change can improve the responsiveness of the imprinted SPR sensor to 3,3′-dichlorobenzidine. The molecularly imprinted polymer gel film was characterized with contact angle measurements, electrochemical impedance spectroscopy, cyclic voltammogram, swelling measurements and atomic force microscopy. The changes of SPR spectroscopy wavenumber shifts revealed that the imprinted gel sensing film can ‘memorize’ the binding of 3,3′-dichlorobenzidine compared to non-imprinted one. The imprinted gel-SPR sensor showed a linear response in the range of 9.0 × 10⁻¹² to 5.0 × 10⁻¹⁰ mol L⁻¹ (R² = 0.9998) for the detection of 3,3′-dichlorobenzidine, and it also exhibited high selectivity to 3,3′-dichlorobenzidine compared to its structurally related analogues. We calculated the detection limits to be 0.471 ng L⁻¹ for tap water and 0.772 ng kg⁻¹ for soil based on a signal to noise ratio of 3. The method showed good recoveries and precision for the samples spiked with 3,3′-dichlorobenzidine. This suggest that the imprinted gel-SPR sensing method can be used as a promising alternative for the detection of 3,3′-dichlorobenzidine.     

Surface plasmon resonance immunosensor for bacteria detection

This work describes an approach for the development of two bacteria biosensors based on surface plasmon resonance (SPR) technique. The first biosensor was based on functionalized gold substrate and the second one on immobilized gold nanoparticles. For the first biosensor, the gold substrate was functionalized with acid-thiol using the self-assembled monolayer technique, while the second one was functionalized with gold nanoparticles immobilized on modified gold substrate. A polyclonal anti-Escherichia coli antibody was immobilized for specific (E. coli) and non-specific (Lactobacillus) bacteria detection. Detection limit with a good reproducibility of 10⁴ and 10³ cfu mL⁻¹ of E. coli bacteria has been obtained for the first biosensor and for the second one respectively. A refractive index variation below 5 × 10⁻³ due to bacteria adsorption is able to be detected. The refractive index of the multilayer structure and of the E. coli bacteria layer was estimated with a modeling software.     

Fluorescence detection of total count of Escherichia coli and Staphylococcus aureus on water-soluble CdSe quantum dots coupled with bacteria

The aim of this paper was to demonstrate a fluorescence measurement method for rapid detection of two bacterial count by using water-soluble quantum dots (QDs) as a fluorescence marker, and spectrofluorometer acted as detection apparatus, while Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were as detection target bacteria. Highly luminescent water-soluble CdSe QDs were first prepared by using thioglycolic acid (TGA) as a ligand, and were then covalently coupled with target bacteria. The bacterial cell images were obtained using fluorescence microscopy. Our results showed that CdSe QDs prepared in water phase were highly luminescent, stable, and successfully conjugated with E. coli and S. aureus. The fluorescence method could detect 10²-10⁷ CFU/mL total count of E. coli and S. aureus in 1-2 h and the low detection limit is 10² CFU/mL. A linear relationship of the fluorescence peak intensity and log total count of E. coli and S. aureus have been established using the equation Y = 118.68X − 141.75 (r = 0.9907).     

Analysis of Strychnos alkaloids in traditional Chinese medicines with improved sensitivity by sweeping micellar electrokinetic chromatography

A new amperometric method was developed for rapid detection of Escherichia coli (E. coli) density using a bienzyme biosensor. The bienzyme biosensor was fabricated based on the covalent immobilization of laccase and horseradish peroxidase (HRP) at indium tin oxide (ITO) electrode by (3-aminopropyl) triethoxysilane (APTES) monolayer. The bienzyme biosensor showed a high sensitivity in determination of the polyphenolic compounds, which was microbially generated from the salicylic acid (SA) added into the culture medium during the course of E. coli metabolism. Since the amount of polyphenolic compounds depends on E. coli density, the bienzyme biosensor was applied for the rapid and high sensitive detection of E. coli density after the E. coli solution was incubated in culture medium with salicylic acid for 2.5 h at 37 °C. By chronoamperometry, the amplified response current was obtained at the bienzyme biosensor, due to the substrate recycling of the polyphenolic compounds driven by bienzyme-catalyzed oxidation and electrochemical reduction. The amplified response current at the biosensor was linear with the E. coli density ranging from 1.6 × 10³ to 1.0 × 10⁷ cells/mL. The bienzyme biosensor could detect the E. coli density with a detection limit of 9.7 × 10² cells/mL within 3 h.     

A surface plasmon resonance probe without optical fibers as a portable sensing device

A surface plasmon resonance (SPR) sensor integrating a small sensor probe, a laser emission diode, a photo detector, and a polarizer was developed as a portable sensing device. The sensor probe was made with a glass cylinder, 50 mm long and 1.5 mm in diameter, that was connected directly to a beam splitter without optical fibers. The SPR spectrum obtained with this probe system showed a 10% reflectivity minimum at 690 nm. Shifts of the SPR spectrum induced by refractive index (RI) changes in the sample were measured by detecting the reflection light intensity at 670 nm. When the sensitivity was compared using a BIAcore™ SPR instrument, the lowest sensor response of 1 mV observed with the SPR probe system coincided with 1.4 × 10⁻⁶ of the RI changes. The RI resolution of the SPR probe was estimated with experimentally evaluated noise on the signal, and, consequently, it was concluded that the RI resolution was 1.2 × 10⁻⁵. Moreover, immunoreaction was demonstrated with adsorbed bovine serum albumin (BSA) and anti-BSA antibody as an analyte. As a result, 50 ng mL⁻¹ of the lower detection limit was estimated.     

Colorimetric detection of iron ions (III) based on the highly sensitive plasmonic response of the N-acetyl-l-cysteine-stabilized silver nanoparticles

We report here a facile colorimetric sensor based on the N-acetyl-l-cysteine (NALC)-stabilized Ag nanoparticles (NALC–Ag NPs) for detection of Fe³⁺ ions in aqueous solution. The Ag NPs with an average diameter of 6.55 ± 1.0 nm are successfully synthesized through a simple method using sodium borohydride as reducing agent and N-acetyl-l-cysteine as protecting ligand. The synthesized silver nanoparticles show a strong surface plasmon resonance (SPR) around 400 nm and the SPR intensity decreases with the increasing of Fe³⁺ concentration in aqueous solution. Based on the linear relationship between SPR intensity and concentration of Fe³⁺ ions, the as-synthesized water-soluble silver nanoparticles can be used for the sensitive and selective detection of Fe³⁺ ions in water with a linear range from 80 nM to 80 μM and a detection limit of 80 nM. On the basis of the experimental results, a new detection mechanism of oxidation–reduction reaction between Ag NPs and Fe³⁺ ions is proposed, which is different from previously reported mechanisms. Moreover, the NALC–Ag NPs could be applied to the detection of Fe³⁺ ions in real environmental water samples.     

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin–strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10⁻¹¹ M to 1 × 10⁻⁶ M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.     

Quantitation of intracellular recombinant human superoxide dismutase using surface plasmon resonance

An immunosensor assay for the quantitation of intracellular recombinant human superoxide dismutase (rhSOD) in Escherichia coli cultivations based on detection with surface plasmon resoance (SPR) is described. A monoclonal antibody for rhSOD was immobilized on a SPR dextran gold chip. Bacterial samples were sonicated and centrifugated prior to injection over the antibody chip for SPR detection. The assay time was 7 min and allowed quantitation in the range of 1-64 nM SOD in lysate samples with a precision of 1.1-3.4%. The assay was applied to monitor the concentration of rhSOD during E. coli bioreactor cultivations where the rhSOD production was induced by iso-propyl-b-d-thiogalactoside (IPTG). The assay allowed accurate monitoring of the production of rhSOD where the important phases in the product formation were possible to see. The report also discusses influence from sample preparation, SPR selectivity and sensitivity and quantitation limits. The assay proved to be fast, sensitive and accurate with low background effects from the dextran matrix of the SPR chip.     

Rapid detection of Escherichia coli O157:H7 spiked into food matrices

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 10³ cells mL⁻¹ were detected in buffer in less than 30 min. Slight losses of sensitivity (1-5 × 10⁻⁴ cell mL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).     

Lectin sensitized anisotropic silver nanoparticles for detection of some bacteria

A method of bacteria detection by sensitized anisotropic silver nanoparticles is presented. Anisotropic silver nanoparticles with two bands of surface plasmon resonance (SPR) are prepared and sensitized with potato lectin. These nanoparticles are able to detect three bacterial species: Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The interaction of these bacteria with such nanoparticles induces drastic changes in optical spectra of nanoparticles that are correlated with bacteria titer. The maximal sensitivity is observed for S. aureus (up to 1.5 × 10⁴ mL⁻¹).     

Sensitive and simple detection of Escherichia coli strain based on time-resolved fluorescence DNA hybridization assay

A two-probe tandem DNA hybridization assay based on time-resolved fluorescence was employed to detect Escherichia coli strain. The amino modified capture probe was covalently immobilized on the common glass slide surface. The Eu(TTA)₃(5-NH₂-phen) with the characteristics of long lifetime and intense luminescence was labeled with reporter probe. The original extracted DNA samples without the purification and amplification process were directly used in the hybridization assay. The concentration of capture probe, hybridization temperature, hybridization and washing time were optimized. The detection limit is about 1.49 × 10³ CFU mL⁻¹E. coli cells, which is comparable to the value of most microbiology methods. The proposed method has the advantages of easy operation, satisfactory sensitivity and specificity, which can provide a promising technique for monitoring the microorganisms.     

Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay

A surface plasmon resonance immunoassay has been developed to determine human growth hormone (hGH) directly and without pre-treatment in human serum samples. A binding inhibition immunoassay was employed. Antibody concentration, assay buffer and regeneration solution have been optimized in order to reach the best performance and the lower non-specific binding of the matrix components to the sensor surface. The lowest detection limit was 6 ng/mL, with a working range covering the physiological range. Reproducibility of the assay was excellent with both intra-assay and inter-assay relative standard deviations <5%, while a variation of 2.19% was obtained employing different sensor chips. Reutilization of the sensor surface allows its continuous use over 50 measurements with a signal drop <20%. The SPR immunoassay results were validated using enzyme-linked immunosorbent assay (ELISA) showing an excellent correlation (R² = 0.985). A portable and fully automated system (Sensia SL) was employed in this work. This is the first SPR biosensor assay capable of detecting relevant concentrations of a clinical analyte in serum. This study shows the potentials of this device as a diagnostic tool for the detection of multiple clinical analytes.     

A gold nanoparticle labeling strategy for the sensitive kinetic assay of the carbamate-acetylcholinesterase interaction by surface plasmon resonance

The article presents a novel strategy for a sensitive investigation of the interaction between acetylcholinesterase (AChE) and its small molecular carbamate inhibitors. Two carbamate inhibitors with different ether linkages and the terminal lipoate were synthesized and labeled with gold nanoparticles (AuNPs). With the signal amplification of AuNPs, the specific interactions between the AuNPs labeled carbamate inhibitors (ALC1 and ALC2) and the immobilized AChE on sensor chip surface were readily examined. The detection sensitivities of ALC1 and ALC2 were 176 and 121 m°/nM, respectively, with the detection limits of 7.0 and 12 pM at a signal-to-noise ratio of 3. The association/dissociation constants for the binding interaction between carbamate inhibitors and AChE were reported for the first time. The affinity constants were estimated to be 3.13 × 10⁶ and 6.39 × 10⁵ M⁻¹ for ALC1 and ALC2 respectively. This AuNPs labeling strategy is versatile and may be applicable for the direct or competitive SPR kinetic assay of the interaction between small molecule inhibitors and their target proteins with a high sensitivity.     

High power impulse magnetron sputtering (HIPIMS) and traditional pulsed sputtering (DCMSP) Ag-surfaces leading to E. coli inactivation

This study addresses the high power impulse magnetron sputtering (HIPIMS) deposition of Ag-nanoparticle films on polyester and the comparison with films deposited by direct current pulsed magnetron sputtering (DCMSP). The first evidence is presented for the Escherichia coli bacterial inactivation by HIPIMS sputtered polyester compared to Ag-polyester sputtered by DCMSP. HIPIMS layers were significantly thinner than the DCMSP sputtered layers needing a much lower Ag-loading to inactivate E. coli within the same time scale. The Ag-nanoparticle films sputtered by DCMSP at 300 mA for 160 s was observed to inactivate completely E. coli within 2 h having a content of 0.205% Ag wt%/polyester wt%. HIPIMS-sputtered at 5 A for 75 s led to complete E. coli bacterial inactivation also within 2 h having a content Ag 0.031% Ag wt%/polyester wt%. The atomic rate of deposition with DCMSP is 6.2 × 10¹⁵ atoms Ag/cm² s while with HPIMS this rate was 2.7 × 10¹⁵ atoms Ag/cm² s. The degree of ionization of Ag⁺/Ag²⁺ and Ar⁺/Ar²⁺ was proportional to the target current applied during HIPIMS-sputtering as determined by mass spectroscopy. These experiments reveal significant differences at the higher end of the currents applied during HIPIMS sputtering as illustrated by the ion-flux composition. X-ray photoelectron spectroscopy (XPS) was used to determine the surface atomic concentration of O, Ag, and C on the Ag-polyester. These surface atomic concentrations were followed during the E. coli inactivation time providing the evidence for the E. coli oxidation on the Ag-polyester. X-ray diffraction shows Ag-metallic character for DCMSP sputtered samples for longer times compared to the Ag-clusters sputtered by HIPIMS leading to Ag-clusters aggregates. Ag-nanoparticle films on polyester sputtered by HIPIMS contain less Ag and are thinner compared to Ag-nanoparticle films sputtered by DCMSP.     

Molecular beacons: a real-time polymerase chain reaction assay for detecting Escherichia coli from fresh produce and water

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >10¹ CFU mL⁻¹ (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples.     

Development of a sensitive detection method of cancer biomarkers in human serum (75%) using a quartz crystal microbalance sensor and nanoparticles amplification system

A simple and sensitive sensor method for cancer biomarkers [prostate specific antigen (PSA) and PSA-alpha 1-antichymotrypsin (ACT) complex] analysis was developed, to be applied directly with human serum (75%) by using antibody modified quartz crystal microbalance sensor and nanoparticles amplification system. A QCM sensor chip consisting of two sensing array enabling the measurement of an active and control binding events simultaneously on the sensor surface was used in this work. The performance of the assay and the sensor was first optimised and characterised in pure buffer conditions before applying to serum samples. Extensive interference to the QCM signal was observed upon the analysis of serum. Different buffer systems were then formulated and tested for the reduction of the non-specific binding of sera proteins on the sensor surface. A PBS buffer containing 200 μg mL⁻¹ BSA, 0.5 M NaCl, 500 μg mL⁻¹ dextran and 0.5% Tween 20, was then selected which eliminated the interfering signal by 98% and enabled the biomarker detection assay to be performed in 75% human serum. By using Au nanoparticles to enhance the QCM sensor signal, a limit of detection of 0.29 ng mL⁻¹ PSA and PSA-ACT complex (in 75% serum) with a linear dynamic detection range up to 150 ng mL⁻¹ was obtained. With the achieved detection limit in serum samples, the developed QCM assay shows a promising technology for cancer biomarker analysis in patient samples.