Smartphone based dual mode in situ detection of viability of bacteria using Ag nanorods array.
Gahlaut Shashank K,Kalyani Neeti,Sharan C,Mishra Prashant,Singh J P
Biosensors & bioelectronics
The in-situ and rapid detection of live and dead bacteria is essential for human and environmental care. It has become one of the biggest needs in the biological and medical sciences to prevent infectious diseases, which usually occur in hospitals and field clinics. In the current scenario, antibiotic resistance is one of the severe public health problems, which requires a quick and efficient solution. Here, we report a facile sensitive, portable, user-friendly, cost-effective and time saving approach for detection of live, dead and drug-resistant bacteria. The endogenous HS evolution was targeted to differentiate between live and dead as well as antibiotic resistant bacteria. The silver nanorods (AgNRs) arrays sensors were fabricated by glancing angle deposition technique. The colorimetric and water wettability features of as-synthesized AgNRs are found to be highly sensitive and selective for HS. E. coli. P. aeruginosa, B. subtilis and S. aureus were used as a model organism in this study. All the bacteria were found to produce HS by their metabolism process. In order to detect the antibiotic resistant E. coli were grown in the presence of different concentration of ampicillin in Luria broth. A drastic visible change in color as well as wetting of AgNRs array was observed. To make the technique easy, a user-friendly and field deployable mobile app 'Colorimetric Detector' was developed. This technique takes only 4-6 h whereas the conventional methods need around 24 h for the same. This dual mode facile and, inexpensive method can be easily scaled up in the field of diagnostics.
In situ formation of gold nanoparticles in polymer inclusion membrane: Application as platform in a label-free potentiometric immunosensor for Salmonella typhimurium detection.
Silva Nádia F D,Magalhães Júlia M C S,Barroso M Fátima,Oliva-Teles Teresa,Freire Cristina,Delerue-Matos Cristina
Polymeric ion selective electrodes are highly sensitive to changes in zero current ion flow and this offers a route to signal amplification in label-free potentiometric immunosensors. In this work, a label-free potentiometric immunosensor toward Salmonella typhimurium (ST) assembled in a home-made pipette-tip electrode is described. The signal-output amplification was implemented on a gold nanoparticle polymer inclusion membrane (AuNPs-PIM) which was used as sensing platform and for antibody immobilization. Additionally, a marker ion was used to detect the antibody-antigen binding event at the electrode surface. The immunosensor construction was performed in several steps: i) gold salt ions extraction in PVC membrane; ii) AuNPs formation using NaEDTA as reduction agent; iii) antibody anti-Salmonella conjugation on AuNPs-PIM in pipette-tip electrodes. The potential shift observed in potentiometric measurements was derived simply from the blocking effect in the ionic flux caused by antigen-antibody conjugation, without no extra steps, mimetizing the ion-channel sensors. A detection limit of 6 cells mL was attained. As proof-of-concept, recovery studies were performed in spiked commercial apple juice samples with success. Due to the simplicity of use, the appealing cost of equipment and sensor production and being able to provide a quick analytical response (less than 1 h for a complete assay, including sample preparation for analysis), this scheme represents a good prototype device for the detection of foodborne pathogens like ST or other immune-responsive bacteria.
Ultrasensitive electrochemical detection of Mycobacterium tuberculosis IS6110 fragment using gold nanoparticles decorated fullerene nanoparticles/nitrogen-doped graphene nanosheet as signal tags.
Bai Lijuan,Chen Yuhan,Liu Xinzhu,Zhou Jing,Cao Jun,Hou Liang,Guo Shuliang
Analytica chimica acta
Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains the top fatal infection continuing to threat public health, and the present detection method for MTB is facing great challenges with the global TB burden. In response to this issue, a novel electrochemical DNA biosensor was developed for detecting the IS6110 fragment within MTB. For the first time, the nanohybrid of gold nanoparticles decorated fullerene nanoparticles/nitrogen-doped graphene nanosheet (Au-nano-C/NGS) directly served as a new signal tag to generate signal response without additional redox molecules and subsequently labeled with signal probes (SPs) to form tracer label to achieve signal amplification. Additionally, a biotin-avidin system was introduced to immobilize abundant capture probes (CPs), further improving the sensitivity of the proposed biosensor. After a typical sandwich hybridization, the proposed electrochemical DNA biosensor was incubated with tetraoctylammonium bromide (TOAB), which was used as a booster to induce the intrinsic redox activity of the tracer label, resulting in a discriminating current response. The proposed electrochemical DNA biosensor shows a broad linear range for MTB determination from 10 fM to 10 nM with a low limit of detection (LOD) of 3 fM. In addition, this proposed biosensor not only distinguishes mismatched DNA sequence, but also differentiates MTB from other pathogenic agents. More importantly, it has been preliminarily applied in clinical detection and displayed excellent ability to identify the PCR products of clinical samples. There is great potential for this developed method to be used in early diagnosis and monitor of TB.
Ultrasensitive dual-color rapid lateral flow immunoassay via gold nanoparticles with two different morphologies for the serodiagnosis of human brucellosis.
Zhu Mingsong,Zhang Jie,Cao Junjun,Ma Jifu,Li Xiangru,Shi Feng
Analytical and bioanalytical chemistry
Lateral flow immunoassays (LFIAs) are popular because they are rapid, convenient, stable, low cost, and easy to read. However, conventional LFIAs based on gold nanoparticles lack sensitivity, which hinders their widespread use. Here, we prepared durian-like gold nanoparticles (GNDs) and labeled them with staphylococcal protein A to detect brucella antibody. Then, the analytical performances of GNDs and gold nanospheres (GNSs) with the same diameter were compared. It was found that the sensitivity of GNDs was five to ten times higher than that of GNSs. The nonspherical morphologies of the nanoparticles greatly increased the sensitivity of the LFIA. On the basis of GNDs and GNSs, we developed an ultrasensitive dual-color brucellosis LFIA. GNSs labeled with streptavidin were used to demonstrate the control line. This dual-color LFIA had a diagnostic sensitivity and specificity of 100%. Human standard Brucella-positive serum (containing brucella antibody at 4000 IU/mL) could be detected in this system even for a dilution factor of 10. The detection limit was 0.04 IU/mL. This is two orders of magnitude better than conventional LFIA strips (detection limit 4 IU/mL). This dual-color LFIA contains all components of a conventional LFIA with no additional processing steps or reagents. It can detect antibodies in serum, plasma, and even whole blood without sample pretreatment or blood filtration pads. Both types of nanoparticles were synthesized in a simple and low-cost manner. This suggests that it will have utility for the early diagnosis of brucellosis and other diseases. Graphical abstract.
Spectrophotometric, colorimetric and visually detection of Pseudomonas aeruginosa ETA gene based gold nanoparticles DNA probe and endonuclease enzyme.
Amini Bahram,Kamali Mehdi,Salouti Mojtaba,Yaghmaei Parichehreh
Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy
Colorimetric DNA detection is preferred over other methods for clinical molecular diagnosis because it does not require expensive equipment. In the present study, the colorimetric method based on gold nanoparticles (GNPs) and endonuclease enzyme was used for the detection of P. aeruginosa ETA gene. Firstly, the primers and probe for P. aeruginosa exotoxin A (ETA) gene were designed and checked for specificity by the PCR method. Then, GNPs were synthesized using the citrate reduction method and conjugated with the prepared probe to develop the new nano-biosensor. Next, the extracted target DNA of the bacteria was added to GNP-probe complex to check its efficacy for P. aeruginosa ETA gene diagnosis. A decrease in absorbance was seen when GNP-probe-target DNA cleaved into the small fragments of BamHI endonuclease due to the weakened electrostatic interaction between GNPs and the shortened DNA. The right shift of the absorbance peak from 530 to 562nm occurred after adding the endonuclease. It was measured using a UV-VIS absorption spectroscopy that indicates the existence of the P. aeruginosa ETA gene. Sensitivity was determined in the presence of different concentrations of target DNA of P. aeruginosa. The results obtained from the optimized conditions showed that the absorbance value has linear correlation with concentration of target DNA (R: 0.9850) in the range of 10-50ngmL with the limit detection of 9.899ngmL. Thus, the specificity of the new method for detection of P. aeruginosa was established in comparison with other bacteria. Additionally, the designed assay was quantitatively applied to detect the P. aeruginosa ETA gene from 10 to 10CFUmL in real samples with a detection limit of 320CFUmL.
Dual-Recognition Förster Resonance Energy Transfer Based Platform for One-Step Sensitive Detection of Pathogenic Bacteria Using Fluorescent Vancomycin-Gold Nanoclusters and Aptamer-Gold Nanoparticles.
Yu Mengqun,Wang Hong,Fu Fei,Li Linyao,Li Jing,Li Gan,Song Yang,Swihart Mark T,Song Erqun
The effective monitoring, identification, and quantification of pathogenic bacteria is essential for addressing serious public health issues. In this study, we present a universal and facile one-step strategy for sensitive and selective detection of pathogenic bacteria using a dual-molecular affinity-based Förster (fluorescence) resonance energy transfer (FRET) platform based on the recognition of bacterial cell walls by antibiotic and aptamer molecules, respectively. As a proof of concept, Vancomycin (Van) and a nucleic acid aptamer were employed in a model dual-recognition scheme for detecting Staphylococcus aureus (Staph. aureus). Within 30 min, by using Van-functionalized gold nanoclusters and aptamer-modified gold nanoparticles as the energy donor and acceptor, respectively, the FRET signal shows a linear variation with the concentration of Staph. aureus in the range from 20 to 10 cfu/mL with a detection limit of 10 cfu/mL. Other nontarget bacteria showed negative results, demonstrating the good specificity of the approach. When employed to assay Staph. aureus in real samples, the dual-recognition FRET strategy showed recoveries from 99.00% to the 109.75% with relative standard derivations (RSDs) less than 4%. This establishes a universal detection platform for sensitive, specific, and simple pathogenic bacteria detection, which could have great impact in the fields of food/public safety monitoring and infectious disease diagnosis.
Endonuclease controlled aggregation of gold nanoparticles for the ultrasensitive detection of pathogenic bacterial DNA.
McVey Claire,Huang Fumin,Elliott Christopher,Cao Cuong
Biosensors & bioelectronics
The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNAse H enzymatic cleavage of the RNA probe, allowing the target DNA to liberate and hybridize with another RNA probe. This continuously happens until all of the RNA probes are cleaved, leaving the nanoparticles unprotected and thus aggregated upon exposure to a high electrolytic medium. The assay is ultrasensitive, allowing the detection of target DNA at femtomolar level by simple spectroscopic analysis (40.7 fM and 2.45fM as measured by UV-vis and dynamic light scattering (DLS), respectively). The target DNA spiked food matrix (chicken meat) is also successfully detected at a concentration of 1.2pM (by UV-vis) or 18.0fM (by DLS). In addition to the ultra-high sensitivity, the total analysis time of the assay is less than 3h, thus demonstrating its practicality for food analysis.
A fluorescence Nano-biosensors immobilization on Iron (MNPs) and gold (AuNPs) nanoparticles for detection of Shigella spp.
Elahi Narges,Kamali Mehdi,Baghersad Mohammad Hadi,Amini Bahram
Materials science & engineering. C, Materials for biological applications
The highly sensitive and specificity detection are very important in diagnosis of foodborne pathogens and prevention of spread diseases. Therefore, in the present study, a highly sensitive fluorescence Nano-biosensors was designed for detection of Shigella species. For achieved this purpose, DNA probes and gold nanoparticles (AuNPs) were designed and synthesized, respectively. Then, two DNA probes as signal reporter were immobilized on surface of AuNPs. On the other hand, Iron nanoparticles (MNPs) were synthesized and modified with SMCC (Sulfosuccinimidyl 4-Nmaleimidomethyl cyclohexane-1- carboxylate). The 3th DNA probe was immobilized on surface of MNPs for separation of target DNA. The MNP-DNA probe and DNA probe-AuNP-fluorescence DNA probe were added to target DNA. The MNP- DNA probe-target DNA-DNA probe-AuNP-fluorescence DNA probe complex was isolated by a magnet. The fluorescence DNA probe was released on surface of AuNPs and the fluorescence intensity was read by fluorescence spectrophotometry. Sensitivity and specificity of designed Nano-biosensor was determined. The results showed that the fluorescence intensity was increased with increasing of target DNA concentration. Linear related between target DNA and fluorescence intensity was observed in 2.3 × 10 up to 2.3 × 10 CFU mL. The linear equation and regression were Y = 1.8 X + 23.4 and R 0.9953. Limit of detection (LOD) were determined 90 CFUmL. The specificity of Nano-biosensor in present of other bacteria was confirmed.
Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.
Amini Bahram,Kamali Mehdi,Salouti Mojtaba,Yaghmaei Parichehreh
Biosensors & bioelectronics
Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R=0.9984) between the fluorescent intensity and the target DNA concentration in the samples.
Ultra-sensitive electrochemical detection of bacteremia enabled by redox-active gold nanoparticles (raGNPs) in a nano-sieving microfluidic system (NS-MFS).
Lee Chun-Wei,Chang Hwan-You,Wu Jen-Kuei,Tseng Fan-Gang
Biosensors & bioelectronics
Early diagnosis of bacterial infections is crucial to improving survival rates by enabling treatment with appropriate antibiotics within the first few hours of infection. This paper presents a highly sensitive amperometric biosensor for the detection of several pathogenic bacterial cells in blood plasma around 30 min. The proposed device is based on an electropolymerized self-assembled layer on gold nanoparticles operated in a portable nano-sieving microfluidic system (NS-MFS). The redox-active gold nanoparticles (raGNPs) enhanced the electrical conductivity and provided a greater number of electrochemically active molecules for sensing, while improving resistance to the fouling of sensors by oxidation products in blood plasma. The detection limit of the device has been shown to reach 10 CFU/mL for Pseudomonas aeruginosa and Staphylococcus aureus spiked in plasma. The dynamic range of the sensing system falls between 10 and 10 CFU/mL in a buffer solution by cyclic voltammetry (CV) measurements. The results demonstrated that the raGNPs/NS-MFS can successful detect P. aeruginosa and S. aureus in human plasma, and is very useful for the diagnosis of bacteremia from clinical samples.
Rapid Colorimetric Detection of Bacterial Species through the Capture of Gold Nanoparticles by Chimeric Phages.
Peng Huan,Chen Irene A
Rapid, inexpensive, and sensitive detection of bacterial pathogens is an important goal for several aspects of human health and safety. We present a simple strategy for detecting a variety of bacterial species based on the interaction between bacterial cells and the viruses that infect them (phages). We engineer phage M13 to display the receptor-binding protein from a phage that naturally targets the desired bacteria. Thiolation of the engineered phages allows the binding of gold nanoparticles, which aggregate on the phages and act as a signal amplifier, resulting in a visible color change due to alteration of surface plasmon resonance properties. We demonstrate the detection of two strains of Escherichia coli, the human pathogens Pseudomonas aeruginosa and Vibrio cholerae, and two strains of the plant pathogen Xanthomonas campestris. The assay can detect ∼100 cells with no cross-reactivity found among the Gram-negative bacterial species tested here. The assay can be performed in less than an hour and is robust to different media, including seawater and human serum. This strategy combines highly evolved biological materials with the optical properties of gold nanoparticles to achieve the simple, sensitive, and specific detection of bacterial species.
Detection of Pyocyanin Using a New Biodegradable SERS Biosensor Fabricated Using Gold Coated Zein Nanostructures Further Decorated with Gold Nanoparticles.
Jia Fei,Barber Emma,Turasan Hazal,Seo Sujin,Dai Ruitong,Liu Logan,Li Xingmin,Bhunia Arun K,Kokini Jozef L
Journal of agricultural and food chemistry
In this paper, a biodegradable gold coated zein film surface enhanced Raman spectroscopy (SERS) platform, with gold nanoparticles (AuNPs) deposited on the surface to further enhance the Raman signal, was used to detect pyocyanin (PYO), the toxin secreted by Pseudomonas aeruginosa. An inverted pyramid structure imprinted on a zein film and gold coated during the transfer process was further improved with the deposition and fixing of gold nanoparticles, which resulted in enhancement of the SERS signal by approximately a decade. This new platform served as a lab-on-a-chip sensor to enable the sensitive and rapid detection of PYO in drinking water. The size, distribution, and morphology of the zein film nanostructures including the presence and distribution of gold nanoparticles were characterized by scanning electron microscopy (SEM). The new zein-based platform has the advantage of being largely biodegradable compared with commercial silicon- or glass-based platforms. The limit of detection for PYO using the newly developed zein-based SERS sensor platform was calculated as 25 μM, considerably lower than the concentration of PYO in the blood of people with cystic fibrosis which has been reported to be 70 μM.
Surface-enhanced Raman spectroscopic single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles.
Wu Shijia,Duan Nuo,Shen Mofei,Wang Jing,Wang Zhouping
A method is described for single-step detection of V. parahaemolyticus in seafood via aptamer-based SERS. A gold-coated polydimethylsiloxane (PDMS) film was used for the enhancement of Raman scattering. The Raman reporter 4-mercaptobenzoic acid was linked to aptamer modified gold nanoparticles (AuNPs) served as a signalling probe. The negatively charged signalling probe was assembled onto the cysteamine-modified Au-PDMS film through electrostatic adsorption. On addition of V. parahaemolyticus, it will be bound by the aptamer as a biorecognition element, and this leads to the dissociation of the signalling probe from the Au-PDMS film. Hence, the Raman signal (at 1592 cm) decreases. The assay has a wide linear response that covers the 1.2 × 10 to 1.2 × 10 cfu·mL V. parahaemolyticus concentration range. The detection limit is 12 cfu·mL. The method was successfully applied to the determination of V. parahaemolyticus in oyster and salmon samples. Graphical abstract Schematic presentation of a surface-enhanced Raman spectroscopic method for single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles. This solid substrate simplified the analysis procedures and enhanced the sensitivity.
Colorimetric Immunoassay for Rapid Detection of Vibrio parahemolyticus Based on Mn Mediates the Assembly of Gold Nanoparticles.
Fu Kaiyue,Zheng Yan,Li Juan,Liu Yushen,Pang Bo,Song Xiuling,Xu Kun,Wang Juan,Zhao Chao
Journal of agricultural and food chemistry
Vibrio parahemolyticus ( V. parahemolyticus) is an important food-borne pathogen that causes food poisoning and acute gastroenteritis in humans. Herein, a novel colorimetric immunoassay was presented for rapid detection of V. parahemolyticus using gold nanoparticles (18.1 nm diameter) as chromogenic substrate, whose combination of a magnetic bead-based sandwich immunoassay and an optical sensing system via Mn ions mediated aggregation of gold nanoparticles. MnO nanoparticles coated with polyclonal IgG antibodies (7.8 nm diameter) are used to recognize the target and can be etched to generate manganese ions by ascorbic acid. A color change ranging from red to purple to blue can be easily discerned by bare eye, corresponding to V. parahemolyticus concentration in the range between 10 and 10 cfu·mL. The proposed method possesses high specificity with a limit of detection of 10 cfu·mL and was successfully applied to determination of V. parahemolyticus in oyster samples without pre-enrichment. In our perception, it shows promise in rapid instrumental and on-site visual detection of V. parahemolyticus.
Gold nanoparticles-based multifunctional nanoconjugates for highly sensitive and enzyme-free detection of E.coli K12.
Zou Yongjin,Liang Jing,She Zhe,Kraatz Heinz-Bernhard
Immobilization of proteins on a biocompatible conductive interface is highly desirable for the fabrication of biosensors. In this study, a nanocomposite has been prepared by assembling well-distributed gold nanoparticles (AuNPs) on the surface of a polypyrrole-reduced graphene oxide (PPy-rGO) composite through electrostatic adsorption. This serves as a platform for immobilization of a capture antibody, which was conjugated onto the ferrocene doped polypyrrole-gold nanoparticles (PPy@Fc/AuNPs) composite. The design and performance of the biosensor was tested against detection of a whole-cell bacteria E. coli K12. This nanocomposite has a high surface area, good conductivity and biocompatibility, which is shown to be very suitable for enzyme-free detection of this bacteria. Results show excellent analytical performance with a linear range from 1.0 × 10 to 1.0 × 10 CFU mL and a low detection limit of 10 CFU mL. The sensor has high selectivity, excellent reproducibility, and good stability.
A simple gold nanoparticle probes assay for identification of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex from clinical specimens.
Soo Po-Chi,Horng Yu-Tze,Chang Kai-Chih,Wang Jann-Yuan,Hsueh Po-Ren,Chuang Chun-Yu,Lu Chia-Chen,Lai Hsin-Chih
Molecular and cellular probes
We had previously developed a nested polymerase chain reaction (PCR)-immunochromatography test (ICT) for identification of Mycobacterium tuberculosis (MTB) and differentiation of MTB from other members of M. tuberculosis complex (MTBC) from clinical sputum samples (Soo P.C. et al., Journal of Microbiological Methods. 2006, 66(3):440-8.). To further improve the detection flexibility, simplicity and efficiency, and reduce the cost, in this study, an alternative molecular diagnosis assay that utilizes gold nanoparticles derivatized with thiol modified oligonucleotides was developed. The gold nanoparticles probes, GP-1/GP-2 for IS6110 and GP-3/GP-4 for Rv3618, were designed to specifically hybridize with target DNAs of MTBC and MTB strains, respectively. Efficacy of the gold nanoparticle probes assay was evaluated by directly and simultaneously detecting not only MTBC but also MTB from 600 clinical sputum specimens. Results were compared with traditional culture and biochemical identification methods together with patients' clinical assessments. This assay showed a 96.6% sensitivity and 98.9% specificity towards detection of MTBC, and a 94.7% sensitivity and 99.6% specificity for detection of MTB. In conclusion, the gold nanoparticle probes assay is a simple, rapid, cost-effective and accurate detection system and shows great potential in clinical application of MTBC and MTB detection, especially in developing countries.
The detection of brucellosis antibody in whole serum based on the low-fouling electrochemical immunosensor fabricated with magnetic FeO@Au@PEG@HA nanoparticles.
Lv Shuli,Sheng Jinliang,Zhao Shiyi,Liu Mingchao,Chen Lihua
Biosensors & bioelectronics
It is a novel competitive challenge for electrochemical biosensor to directly, rapidly and ultrasensitively detect the disease markers in the whole serum due to biofouling caused by the complexity of actual samples. In this paper, poly (ethylene glycol) (PEG) and hyaluronic acid (HA) were utilized to modify FeO @Au nanoparticles (NPs). Based on the successfully preparation and characterization of FeO @Au@PEG@HA NPs with TEM, SEM, XRD, FTIR and EDS, respectively, a novel immunosensor of brucellosis with high selectivity, sensitivity and almost perfect protein-resistant properties in various external environments, especially, in complex biological systems was fabricated. More importantly, this immunosensor is capable of assaying brucellosis antibody in 100% serum without suffering from any significant biological interference. In addition, a wide linear response range from 10 g mL to 10 g mL towards antibody in 100% serum and a low limit of detection (LOD) of 0.36 fg mL (3σ, n = 13) are demonstrated, which indicates that this immunosensor has a promising potential in clinical diagnosis.
Rapid identification of Salmonella using Hektoen enteric agar and 16s ribosomal DNA probe-gold nanoparticle immunochromatography assay in clinical faecal specimens.
Yeung C-Y,Liu C-C,Tseng Y-T,Tsai K-C,Hsieh M-A,Chan W-T,Liu H-L,Lee H-C,Hou S-Y
Letters in applied microbiology
UNLABELLED:A rapid identification of Salmonella, one of the most common foodborne pathogens worldwide, in clinical patients can enable better rational managements and prevent further outbreaks. The traditional immunochromatography using antibody-gold nanoparticles (Ab-AuNPs), such as the home pregnancy test, has been used for the Salmonella detection. In this study, we developed a new and rapid method using DNA probe-AuNPs for the detection of 16s ribosomal DNA of Salmonella. To evaluate the proposed method in clinical specimens, we performed a clinical test by identifying 159 stool samples on Hektoen agar containing black or crystalloid colonies using the method and the VITEK 2 system for confirmation. Eighty of the isolates were correctly identified as Salmonella to achieve 100% sensitivity. Seventy-five samples were correctly identified as non-Salmonella spp., but four were incorrectly identified as Salmonella. The specificity was 94·93%. The assay time is about 30 min after the DNA purification. The time-consuming and labour-intense biochemical tests can be replaced. We demonstrated that this assay is a rapid, convenient and cost-effective tool for Salmonella identification of clinical faecal samples, which is worth for further promotion and clinical use. This is the first application of using 16s ribosomal DNA probe-Au-NPs and immunochromatography on clinical samples. SIGNIFICANCE AND IMPACT OF THE STUDY:This is the first application of using 16s ribosomal DNA probe-gold nanoparticles and immunochromatography method on clinical samples with sensitivity 100% and specificity 94·93%. The assay time is about 30 min after the DNA purification. We find this assay a rapid, convenient, sensitive and inexpensive tool for Salmonella identification of clinical faecal samples, which is worth further promotion and clinical use and can replace the traditional time-consuming and labour-intense biochemical tests. The potential benefit of this approach is to develop a rapid point-of-care test that provides results while the patient is still at the doctors' office.
Rapid and sensitive detection of Pseudomonas aeruginosa using multiple cross displacement amplification and gold nanoparticle-based lateral flow biosensor visualization.
Zhao Fan,Niu Lina,Nong Jinqing,Wang Chunmei,Wang Jing,Liu Yan,Gao Naishu,Zhu Xiaoxue,Wu Lei,Hu Shoukui
FEMS microbiology letters
Pseudomonas aeruginosa causes nosocomial infections of burn patients and other immunocompromised individuals, but the conventional diagnosis of P. aeruginosa infection depends on time-consuming culture-based methods. Hence, a simple, fast, sensitive technique for detection of P. aeruginosa using multiple cross displacement amplification (MCDA) and gold nanoparticle-based lateral flow biosensors (LFB) was developed. By using this technique, the reaction could be completed at an optimized constant temperature (67°C) within only 40 min. The reaction product could be detected visually using an LFB, eliminating the need for special equipment. The P. aeruginosa-MCDA-LFB method was highly specific, and accurately distinguished P. aeruginosa from other pathogens. Just 10 fg of genomic DNA template (from pure culture) could be detected. The assay could also detect P. aeruginosa in clinical sputum samples and showed the same sensitivity and specificity as the reference (culture-biochemical) method. In the future, this rapid, simple and accurate P. aeruginosa-MCDA-LFB technique might be applied in clinical practice.
Gold nanoparticle triggered siloxane formation for polymerization based amplification in enzyme free visual immunoassay.
Singh Pargat,Bharti ,Kumar Rajesh,Bhalla Vijayender
Analytica chimica acta
Herein, we report a new signal amplification scheme for quantitative biochemical analysis based on gold nanoparticle (GNPs) catalyzed polymerization of transparent silane solution to milky white and turbid siloxane. Using immunoassay as a proof of concept, GNP labeled immunoprobe was used to bind captured antigen and catalyse the polymerization reaction allowing sensitive biochemical investigation. The polymerization reaction was optimized for standard 96 well polystyrene microtiter plates and we discovered that sodium lactate acts as an enhancer in the polymerization reaction as it reduces detection time to merely 30 min. The sensing strategy was applied to detection and quantification of Salmonella Typhimurium in water and egg samples and the platform showed excellent visibly quantifiable analytical response up to 100 cells mL. Furthermore, clinical utility and potential of the method was validated by detecting Vi capsular polysaccharide (Vi antigen) responsible for typhoidal Salmonellosis in human serum in sandwich format with a detection limit of 1 ng mL. The method serves as the first report towards nanoparticle triggered polymerization for development of rapid and low cost quantitative biochemical assay.
A sensitive colorimetric assay for identification of Acinetobacter baumannii using unmodified gold nanoparticles.
Khalil M A F,Azzazy H M E,Attia A S,Hashem A G M
Journal of applied microbiology
AIMS:Acinetobacter baumannii is a global health problem, which threatens many healthcare settings. The current study aims to develop a detection assay for Ac. baumannii using unmodified gold nanoparticles (AuNPs). METHODS AND RESULTS:Fifty-three Ac. baumannii clinical isolates were collected from Egyptian hospitals. Bacterial isolation and biochemical identification of isolates were carried out followed by DNA extraction using boiling method and PCR amplification of the 23S-16S rRNA intergenic spacer sequences (ITS). AuNPs were synthesized using citrate reduction method. Detection and optimization of Ac. baumannii amplicons using unmodified spherical AuNPs were performed using species-specific DNA oligonucleotide. The nano-gold assay was able to colorimetrically detect and distinguish Ac. baumannii from other Gram-negative bacteria. The turnaround time of the assay is about 2 h including sample treatment and amplification. The assay detection limit is 0·8125 ng of DNA. CONCLUSIONS:The developed colorimetric assay is sensitive, fast and reliable and can be used for identification of Ac. baumannii. SIGNIFICANCE AND IMPACT OF THE STUDY:There is a need to develop robust, rapid, and specific methods for detection of Ac. baumannii isolated from clinical specimens. The developed nanogold assay prototype allows sensitive, specific and rapid detection of amplified DNA of A. baumannii and represents a reliable diagnostic tool to aid routine laboratory identification of this pathogen.
Unmodified gold nanoparticles for direct and rapid detection of Mycobacterium tuberculosis complex.
Hussain Marwa M,Samir Tamer M,Azzazy Hassan M E
OBJECTIVES:This work aims to develop rapid nano-gold assay prototypes for specific detection of Mycobacterium tuberculosis complex (MTBC). DESIGN AND METHODS:Spherical gold nanoparticles (AuNPs, 14nm) were synthesized by citrate reduction method and characterized by spectrophotometry and SEM. MTB 16s rDNA regions were amplified by PCR and amplicons were detected using genus- and species-specific oligotargeters and AuNPs. In a second prototype, MTBC unamplified genomic DNA was directly detected using species-specific oligo-targeters and AuNPs. RESULTS:Detection limits were 1ng for PCR product and 40ng for genomic DNA. The nano-gold prototype detected 45 positive genomic DNA samples which were also positive with automated liquid culture system (BACTEC™ MGIT™) and semi-nested PCR (100% concordance). Following DNA extraction, using standard procedures, the TB nano-gold prototype turnaround time is about 1h. CONCLUSIONS:We have developed nano-gold assay prototype for direct and inexpensive detection of MTBC. The developed prototypes are simple, sensitive, rapid and can substitute PCR-based detection. The developed assay may show potential in the clinical diagnosis of TB especially in developing countries.
Pathogenic Escherichia coli (E. coli) detection through tuned nanoparticles enhancement study.
Satpathy Gargibala,Chandra Goutam Kumar,Manikandan E,Mahapatra D Roy,Umapathy Siva
OBJECTIVE:This study aims to detect pathogenic Escherichia coli (E. coli) bacteria using non-destructive fluorescence microscopy and micro-Raman spectroscopy. RESULTS:Raman vibrational spectroscopy provides additional information regarding biochemical changes at the cellular level. We have used two nanomaterials zinc oxide nanoparticles (ZnO-NPs) and gold nanoparticles (Au-NPs) to detect pathogenic E. coli. The scanning electron microscope (SEM) with energy dispersive X-ray (EDAX) spectroscopy exhibit surface morphology and the elemental composition of the synthesized NPs. The metal NPs are useful contrast agents due to the surface plasmon resonance (SPR) to detect the signal intensity and hence the bacterial cells. The changes due to the interaction between cells and NPs are further correlated to the change in the surface charge and stiffness of the cell surface with the help of the fluorescence microscopic assay. CONCLUSIONS:We conclude that when two E. coli strains (MTCC723 and MTCC443) and NPs are respectively mixed and kept overnight, the growth of bacteria are inhibited by ZnO-NPs due to changes in cell membrane permeability and intracellular metabolic system under fluorescence microscopy. However, SPR possessed Au-NPs result in enhanced fluorescence of both pathogens. In addition, with the help of Raman microscopy and element analysis, significant changes are observed when Au-NPs are added with the two strains as compared to ZnO-NPs due to protein, lipid and DNA/RNA induced conformational changes.
Thiol-Capped Gold Nanoparticle Biosensors for Rapid and Sensitive Visual Colorimetric Detection of Klebsiella pneumoniae.
Ahmadi Sepideh,Kamaladini Hossein,Haddadi Fatemeh,Sharifmoghadam Mohammad Reza
Journal of fluorescence
In the last few years, gold nanoparticle biosensors have been developed for rapid, precise, easy and inexpensive with high specificity and sensitivity detection of human, plant and animal pathogens. Klebsiella pneumoniae serotype K2 is one of the common gram-negative pathogens with high prevalence. Therefore, it is essential to provide the effective and exclusive method to detect the bacteria. Klebsiella pneumoniae serotype K2 strain ATCC9997 genomic DNA was applied to establish the detection protocol either with thiol-capped oligonucleotide probes and gold nanoparticles or polymerase chain reaction based on K2A gene sequence. In the presence of the genomic DNA and oligonucleotide probes, a change in the color of gold nanoparticles and maximum changes in wavelength at 550-650 nm was achieved. In addition, the result showed specificity of 15 × 10 CFU/mL and 9 pg/μL by gold nanoparticles probes. The lower limit of detection obtained by PCR method was 1 pg/μL. Moreover, results demonstrated a great specificity of the designed primers and probes for colorimetric detection assay and PCR. Colorimetric detection using gold nanoparticle probe with advantages such as the lower time required for detection and no need for expensive detection instrumentation compared to the biochemical and molecular methods could be introduced for rapid, accurate detection of the bacteria.