Strip Biosensor Research Articles

Enzyme-free strip biosensor for amplified detection of Pb2+based on a catalytic DNA circuit

A simple and enzyme-free strip biosensor for the amplified detection of Pb(2+) has been constructed based on a catalytic DNA circuit. This assay is ultrasensitive, enabling the visual detection of Pb(2+) concentrations as low as 10 pM without instrumentation.

Nucleic-Acid Based Lateral Flow Strip Biosensor via Competitive Binding for Possible Dengue Detection

A low-cost, simple, rapid and selective nucleic-acid based lateral flow strip biosensor (LFSB) for possible dengue viral RNA detection is described in this study. The detection is based on competitive binding, where gold nanoparticles (AuNPs), with average size of ~10 nm confirmed using UV-Vis, TEM and AFM images, are used as visualizing agents. These are bioconjugated with DNA which competitively binds with its complementary strand either in the sample or in the test line of the LFSB. The detection scheme reduces the number of probes which effectively lowers the cost for the design of the test strip. The whole test took less than five minutes to complete and a red line signifies a negative result, while the absence of the line signifies a positive result. Quantification of the intensity of the red band reveals proportionality of the color to the amount of DNA present in the sample. The visual limit of detection of the LFSB is 10-7 M. It demonstrates selectivity in a blood matrix and selectivity over a syntheticm Influenza. This study brings us closer to an amplification-free, point-of-care method for dengue detection.

A nanoparticle-based sensor for visual detection of multiple mutations

Disposable dipstick-type DNA biosensors in the form of lateral flow strips are particularlyuseful for genotyping in a small laboratory or for field testing due to their simplicity, lowcost and portability. Their unique advantage is that they enable visual detection in minuteswithout the use of instruments. In addition, the dry-reagent format minimizes thepipetting, incubation and washing steps. In this work, we significantly enhance themultiplexing capabilities of lateral flow strip biosensors without compromising theirsimplicity. Multiplex genotyping is carried out by polymerase chain reaction (PCR)followed by a single primer extension reaction for all target alleles, in which a primer isextended and biotin is incorporated only if it is perfectly complementary to the target.Multiallele detection is achieved by multiple test spots on the membrane of thesensor, each comprising a suspension of polystyrene microspheres functionalizedwith capture probes. The products of the primer extension reaction hybridize,through specific sequence tags, to the capture probes and are visualized by usingantibiotin-conjugated gold nanoparticles. This design enables accommodation ofmultiple spots in a small area because the microspheres are trapped in the fibresof the membrane and remain fixed in site without any diffusion. Furthermore,the detectability is improved because the hybrids are exposed on the surfaceof the trapped microspheres rather than inside the pores of the membrane. Wedemonstrate the specificity and performance of the biosensor for multiallele genotyping.

Ultrasensitive nucleic acid biosensor based on enzyme–gold nanoparticle dual label and lateral flow strip biosensor

In this article, we describe an ultrasensitive nucleic acid biosensor (NAB) based on horseradish peroxidase (HRP)–gold nanoparticle (Au-NP) dual labels and lateral flow strip biosensor (LFSB). The results presented here expand on prior work ( Mao et al., 2009a) by optimizing the preparation of HRP–Au-NP–DNA conjugates. It was found that sodium dodecyl sulfate (SDS) and the immobilization sequence of thiolated DNA and HRP on the Au-NP surface played very important roles to improve the sensitivity of the assay. After systematic optimization, the detection limit of current approach is 1000 times lower than that in prior work. Deposition of insoluble enzymatic catalytic product (red colored chromogen) on the captured Au-NPs at the test zone of LFSB offers a dramatic visual enhancement. Combining enzyme catalytic amplification with unique optical properties of Au-NPs, the NAB was capable of detecting of 0.01-pM target DNA without instrumentation. The NAB thus provides a rapid, sensitive, low-cost tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.

Visual Detection of Single-Nucleotide Polymorphism with Hairpin Oligonucleotide-Functionalized Gold Nanoparticles

We report a simple, fast, and sensitive approach for visual detection of single-nucleotide polymorphism (SNP) based on hairpin oligonucleotide-functionalized gold nanoparticle (HO-Au-NP) and lateral flow strip biosensor (LFSB). The results presented here expand on prior work ( Mao , X. , Xu , H. , Zeng , Q. , Zeng , L. , and Liu , G. Chem. Commun. 2009 , 3065-3067 .) by providing new approach to prepare HO-Au-NP conjugates with a deoxyadenosine triphosphate (dATP) blocker, which shortens the preparation time of the conjugates from 50 to 8 h and lowers the detection limit 500 times. A hairpin oligonucleotide modified with a thiol at the 5'-end and a biotin at the 3'-end was conjugated with Au-NP through a self-assembling process. Following a blocking step with dATP, the hairpin structure of HO and dATP embed the biotin groups, and make the biotin groups in close proximity to the Au-NP surface, leading to the biotins being "inactive". The strategy of detecting SNP depends on the unique molecular recognition properties of HO to the perfect-matched DNA and single-base-mismatched DNA to generate different quantities of "active" biotin groups on the Au-NP surface. After hybridization reactions, the Au-NPs associated with the activated biotins are captured on the test zone of LFSB via the specific reaction between the activated biotin and preimmobilized streptavidin. Accumulation of Au-NPs produces the characteristic red bands, enabling visual detection of SNP. The preparations of HO-Au-NP conjugates with dATP and the parameters of assay were optimized systematically, and the abilities of detecting SNP were examined in details. The current approach is capable of discriminating as low as 10 pM of perfect-matched DNA and single-base-mismatched DNA within 25 min without instrumentation. Moreover, the approach provides a lower background and higher selectivity compared to the current molecular beacon-based SNP detection. The protocol should facilitate the simple, fast, and cost-effective screening of important SNPs and could readily find wide applications in molecular diagnosis laboratories and in point-of-care testing (field testing).

Paper Strip Whole Cell Biosensors: A Portable Test for the Semiquantitative Detection of Bacterial Quorum Signaling Molecules

Herein, we report the development of a novel, inexpensive, and portable filter-paper-based strip biosensor for the detection of bacterial quorum sensing signaling molecules, N-acylhomoserine lactones (AHLs). AHLs are generally employed by Gram-negative bacteria for their cell-cell communication to control expression of specialized genes, such as those involved in biofilm formation and production of virulence factors, in a population-density-dependent manner. First, a bacterial cell-based sensing system employing components of AHL-mediated QS regulatory system as recognition elements and beta-galactosidase as the reporter protein was designed and developed. The bacterial-sensing cells were then liquid-dried on strips of filter paper. beta-Galactosidase as the reporter allows for the visual monitoring of the analyte-induced signal when a colorimetric method of detection is applied. The paper strip biosensor was able to detect low AHL concentrations down to 1 x 10(-8) M. Furthermore, it was successfully applied to the detection of AHLs in physiological samples, such as saliva. The filter-paper-based sensing strips could provide reproducible results upon storage at 4 degrees C for at least 3 months. In conclusion, a filter-paper-based strip biosensor was developed that allows for visual, fast, and convenient detection of AHLs in a dose-dependent manner in a test sample. In addition, it does not require expensive equipment or trained personnel and allows ease of transportation and storage. Therefore, we envision that this biosensor will serve as a simple and economical portable field kit for on-site monitoring of AHL in a variety of clinical and environmental samples.

Characterization of polyaniline-coated magnetic nanoparticles for application in a disposable membrane strip biosensor

We investigated the electrical properties of polyaniline-coated magnetic nanoparticles as a signal transducer for application in a disposable membrane strip biosensor. The size of these particles (~100 nm) was investigated by a transmission electron microscope. Electrical properties of these nanoparticles were investigated by four-point probe measurements and I-V measurements. Polyaniline-coated magnetic nanoparticles had a resistivity of 0.385 Ω cm and showed ohmic behavior. Resistance decreased with increasing concentration of polyaniline. We also demonstrated that the resistance decreased with increasing concentration of biotinylated IgG conjugated with these nanoparticles.

Moving Enzyme-Linked ImmunoSorbent Assay to the Point-of-Care Dry-Reagent Strip Biosensors

In this work, we described a point-of-care (POC) dry-reagent strip biosensor (DRSB) based on enzyme tracers and portable strip reader for simple, low-cost and sensitive assay of protein detection in minutes. Horseradish Peroxidase (HRP) and Rabbit IgG (R-IgG) were used as a model system for the demonstration of the proof-of-concept. The sandwich-type immunoreactions were performed on the DRSB and the HRP tracers were captured on the test zone of the biosensor. The excess of HRP tracers were captured on the control zone of the biosensor through the immobilized secondary antibody. Subsequent enzymatic reaction in the presence of the substrate produced insoluble enzymatic products, which deposited on both test and control zones of the DRSB and formed two characteristics blue bands. While qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the intensities of the test zone with a portable “strip reader”. The quantitative response of the optimized DRSB over the range of 0.1-50 ng mL -1 IgG in association with a 10-min assay time is obtained, and the limit of detection is estimated to be 0.05 ng/mL, which is ten times lower than that of the gold nanoparticle (GNP)-based DRSB. The enzyme-based DRSB was used to detect Carcinoembryonic Antigen (CEA) biomarker in human plasma successfully. Such enzyme-based DRSB offers a simple and fast tool for point-of-care protein assay and a potential substituent for the traditional Enzyme-linked Immunosorbent Assay (ELISA).

Aptamer−Nanoparticle Strip Biosensor for Sensitive Detection of Cancer Cells

We report an aptamer-nanoparticle strip biosensor (ANSB) for the rapid, specific, sensitive, and low-cost detection of circulating cancer cells. Known for their high specificity and affinity, aptamers were first selected from live cells by the cell-SELEX (systematic evolution of ligands by exponential enrichment) process. When next combined with the unique optical properties of gold nanoparticles (Au-NPs), ANSBs were prepared on a lateral flow device. Ramos cells were used as a model target cell to demonstrate proof of principle. Under optimal conditions, the ANSB was capable of detecting a minimum of 4000 Ramos cells without instrumentation (visual judgment) and 800 Ramos cells with a portable strip reader within 15 min. Importantly, ANSB has successfully detected Ramos cells in human blood, thus providing a rapid, sensitive, and low-cost quantitative tool for the detection of circulating cancer cells. ANSB therefore shows great promise for in-field and point-of-care cancer diagnosis and therapy.

Molecular beacon-functionalized gold nanoparticles as probes in dry-reagent strip biosensor for DNA analysis

The highly specific molecule recognition properties of molecular beacons (MB) are combined with the unique optical properties of gold nanoparticles (Au-NPs) for the development of a dry-reagent strip-type nucleic acid biosensor (DSNAB) that enables sensitive and low-cost detection of nucleic acid samples within 15 min.

Aptamer-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for Protein Analysis

The highly specific molecular recognition properties of aptamers are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip biosensor that enables qualitative (visual)/quantitative detection of protein within minutes. A model system comprising thrombin as an analyte and a pair of aptamer probes is used to demonstrate the proof-of-concept on the conventional lateral flow test strip. The assay avoids the multiple incubation and washing steps performed in most current aptamer-based protein analyses. Although qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the optical responses of the test zone with a portable "strip reader". The response of the biosensor is linear over the range of 5-100 nM of thrombin with a detection limit of 2.5 nM (S/N = 3). By comparing the analytical performances of the aptamer-based strip biosensor with the antibody-based strip sensor, we can demonstrate that aptamers are equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. The sensor was used successfully for detection of thrombin in human plasma samples. It shows great promise for use of aptamer-functionalized gold nanoparticle probes in dry-reagent strip biosensors for point-of-care or in-field detection of proteins.

Mixed self-assembly of polydiacetylenes for highly specific and sensitive strip biosensors

Polydiacetylenes (PDAs), which possess unique properties that allow them to change color in response to environmental changes such as variations in pH, temperature, and molecular binding, have been widely investigated as signal transducers in biosensor applications. Most PDA-based sensors reported to date have been evaluated largely on the basis of their ability to detect purified samples, however, and their specificity has rarely been tested. In this study, novel PDAs fabricated on polyvinylidene fluoride (PVDF) strips by photoreaction of composite diacetylene self-assemblies were developed as biosensors, and nonspecific binding to off-target biomolecules was assessed. A mixed PDA surface containing biotin and ethanolamide bound the target, i.e., streptavidin, more specifically than did biotin alone. The optimized PDA biosensor exhibited approximately 2850-fold higher selectivity for streptavidin relative to bovine serum albumin controls. A PDA biosensor that was further prepared showed distinctive signals for the urine of diabetic patients compared to urine samples from healthy/non-diabetic person due to the concentration of microalbuminuria. To our knowledge, this is the first strip-type biosensor fabricated with PDAs and the first PDA-based biosensor that can effectively overcome the problem of nonspecific binding.

Strip bioelectrochemical cell for potentiometric measurements fabricated by screen-printing

Miniaturized, planar bioelectrochemical cell dedicated for potentiometric detection of urea manufactured in integrated strip format has been developed. The enzyme electrode and the reference electrode, both based on ruthenium dioxide were fabricated only by means of screen-printing. The application of the thick-film technology results in highly reproducible and cost-effective manufacturing procedure, easily adapted to large-scale production. The developed system of planar electrodes can be applied as a disposable device for drop-on measurements as well as a detector for a multiple use in flow injection analysis. The biosensor is useful for determination of urea in millimolar range of concentrations. The developed strip biosensor is also useful as a disposable device for potentiometric detection of heavy metal ions, which are able to inhibit urease. The utility of the developed devices for determination of urea in urine and serum samples is demonstrated.

Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization.

The highly specific molecular recognition properties of oligonucleotides are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip-type biosensor that enables visual detection of double stranded DNA within minutes. The assay does not require instrumentation and avoids the multiple incubation and washing steps performed in most current assays. Gold nanoparticle reporters with oligo(dT) attached to their surface form an integral part of the strip. Biotinylated PCR products (233 bp or 495 bp) are hybridized (5 min) with a poly(dA)-tailed oligo and applied on the strip, which is then immersed in the appropriate buffer. As the buffer migrates upward, it rehydrates the nanoparticles that are linked to the target DNA through poly(dA)/(dT) hybridization. Capture of the hybrids by immobilized streptavidin in the test zone of the strip generates a characteristic red band. A second red band is formed, by hybridization, in the control zone of the strip to indicate proper test performance. The sensor offers at least 8 times higher detectability than ethidium bromide staining of agarose gels and provides confirmation of the amplified fragments. Quantitative data are obtained by densitometric analysis of the bands. As low as 2 fmol of amplified DNA were detectable by the strip sensor. Also, 500 copies of prostate-specific antigen cDNA were detected by combining PCR and the strip sensor. The sensor was used successfully for detection of hepatitis C virus in plasma samples from 20 patients. The strip detected 16 out of 16 positive samples and gave no signal for 4 samples that were negative for the virus. To our knowledge, this is the first dry-reagent system that makes use of oligonucleotide-conjugated gold nanoparticles as probes.