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Detection of Fragmented Genomic DNA by PCR-Free Piezoelectric Sensing Using a Denaturation Approach
Abstract
Label-free and real-time DNA sequence detection in PCR-amplified DNA samples can now be achieved by different approaches. On the contrary, only few works have been reported dealing with direct sequence detection in nonamplified genomic DNA. Here, a piezoelectric biosensor for direct detection of sequences in nonamplified genomic DNA is described. The system relies on real-time and label-free detection of the hybridization reaction between an immobilized probe and the complementary sequence in solution. The DNA probe is immobilized on the sensing surface (10 MHz quartz crystals), while the complementary sequence is present in the genomic DNA, previously fragmented with restriction enzymes.
... Development of piezoelectric DNA-based biosensor for direct detection of highly repeated sequences using non-amplified genomic DNA has been recently reported. Minnunni et al. [49][50][51] detected satellite 13 DNA from genomic DNA of Bos taurus. They also applied this method for direct detection of single copy gene in non-amplified genomic DNA from Nicotiana glauca tobacco plant. ...
... The thermal plus blocking oligonucleotides gave the frequency change (indecreasing the resonant frequency) higher than only thermal denaturation. The thermal plus blocking oligonucleotides gave the frequency changes higher than the frequency change of thermal denaturation only [50,51,7]. These thermal plus blocking oligonucleotides method could be improved the sensitivity by using mass enhancement (gold nanoparticle) was capped with blocking oligonucleotides by using 20 nm diameter of gold nanoparticle as optimal for the surface immobilization of QCM biosensor and the consequent sensitivity improvement because the hybridization was highest maximum value when the average diameter of nanoparticles was 20 nm and then decreased with the increasing of particle size [53]. ...
Generally, the detection of particular DNA sequence is carried out by gel electrophoresis of DNA fragments amplified by polymerase chain reaction (PCR) using primers which are specific complementary sequence with the chosen region of DNA. Although gel electrophoresis is simple and effective for detection of PCR products, the use of carcinogenic ethidium bromide as a common stain is risky and hazardous. The DNA quartz crystal microbalance (DNA-QCM) lays the groundwork for incorporating the method for rapid DNA analysis. QCM biosensor is an extremely sensitive mass sensor, capable of measuring picogram levels of mass changes. This is a great potential market for simple, cheap, rapid, and quantitative detection of specific genes. QCM requires any non-labeling such as radio isotope, enzyme, and fluorescence tag.
... But this treatment was not enough for non-amplified genomic DNA because of reannealing of bacterial target DNA. Minnuni and colleague [7,18] used the blocking oligonucleotides for blocked the bacterial target DNA after simple thermal treatment. This method increases the efficiency of hybridization between DNA probe and non-amplified genomic DNA. ...
... The thermal plus blocking oligonucleotides capture with avidin (0.2 mg/mL) gave the frequency shift (in decreasing the resonant frequency) higher than thermal denaturation plus blocking primer only. This result shows the similarity with previous studies [7,18] which the thermal plus blocking oligonucleotides gave the frequency shift higher than the frequency shift of thermal denaturation only. ...
This study was focused on establishment of piezoelectric biosensor for direct detection of
Mycobacterium tuberculosis (MTB) in clinical specimens. The quartz crystal immobilized via
3-mercaptopropionic acid (MPA)/avidin/DNA biotinylated probe on gold surface and hybridization of
the DNA target to DNA biotinylated probe. The optimal concentration of MPA, avidin and
5’-biotinylated DNA probe for immobilization of specific DNA probe on gold surface were 15 mM,
0.1 mg/ml and 1.5 μM, respectively. The detection of genomic DNA digestion in the range from 0.5 to
30 μg/ml. The fabricated biosensor was evaluated through an examination of 200 samples. No cross
hybridization were observed against M. avium complex (MAC) and other microorganism. This target
DNA preparation without amplification will reduce time consuming, costs, and the tedious step of
amplification. This study can be extended to develop the new method which is high sensitivity,
specificity, cheap, easy to use, and rapid for detection of MTB in many fields.
... Hybridization with both a capture and a label probe is difficult because long genomic DNA can supercoil, diffuses slowly, and because the rehybridization of the long DNA strands is kinetically favorable. [23,24] The solution to this problem is to fragment the genomic material into many smaller pieces. Restriction enzymes cut double stranded DNA at specific recognition sites, typically 4-8 bp long. ...
... The benefits we observe with blocking agree with similar work on detection of genomic material reported in the literature. [23,25] The combination of fragmentation by restriction enzymes and blocking for the detection of mtDNA is shown in the micrographs in Figure 2. We designed probe sets for a triplex assay for the NADH gene found in human, swine, and bovine mtDNA. Using synthetic ssDNA where the 54-nt target sequence was found in the middle of 104-nt long strand, we verified that our probe sets were appropriate for multiplexed analysis. ...
Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.
... In case of low concentrations, amplification strategies can be applied before the PZ measurements. The direct PZ detection of non-amplified target sequence was reported, too [37]. The fragmentation of the genomic sequence with restrictase, thermal denaturation, and blocking of renaturation using short oligonucleotides was critical for successful performance. ...
The three decades of experience with piezoelectric devices applied in the field of bioanalytical chemistry are shared. After introduction to principles and suitable measuring approaches, active and passive methods based on oscillators and impedance analysis, respectively, the focus is directed towards biosensing approaches. Immunosensing examples are provided, followed by other affinity sensing approaches based on hybridization of nucleic acids, aptamers, monitoring of enzyme activities, and detection of pathogenic microbes. The combination of piezosensors with cell lines and testing of drugs is highlighted, including mechanically active cells. The combination of piezosensors with other measuring techniques providing original hybrid devices is briefly discussed.
Graphical Abstract
... DNA biosensors based on nucleic acid recognition have applications such as in electrophoresis analysis of amplified DNA. The applications of DNA-based biosensor analysis extend to the field of food control, process control of raw materials, and traceability in industrial processing plants, and in the field of food control, not only for raw materials but also for process control and traceability in industrial processing plants (Minunni et al. 2005;Mannelli et al. 2003;Bogani et al. 2008). Label-free piezoelectric DNA biosensors present adequate specificity and high sensitivity, allowing rapid and real-time control of DNA hybridisation (Lucarelli et al. 2008;Wu et al. 2007;Sun et al. 2006). ...
Nanotechnology is an emerging technological and scientific break through that can transform agricultural sectors by providing novel tools for the molecular detection of biotic and abiotic stress, and the rapid detection of phytopathogenic diseases.
... DNA biosensors based on nucleic acid recognition have applications such as in electrophoresis analysis of amplified DNA. The applications of DNA-based biosensor analysis extend to the field of food control, process control of raw materials, and traceability in industrial processing plants, and in the field of food control, not only for raw materials but also for process control and traceability in industrial processing plants (Minunni et al. 2005;Mannelli et al. 2003;Bogani et al. 2008). Label-free piezoelectric DNA biosensors present adequate specificity and high sensitivity, allowing rapid and real-time control of DNA hybridisation (Lucarelli et al. 2008;Wu et al. 2007;. ...
Barley is regarded as the globe’s fourth major cereal crop. A variety of airborne, seedborne, and soilborne infective agents attack barley, causing a variety of barley diseases and substantial losses in agricultural output. Brown and yellow rusts, smut, net blotches, spot blotches, barley yellow dwarf, and molya disease are among the most serious diseases. In general, employing integrated disease management approaches is the best way to handle barley diseases. Growing resistant or tolerant varieties with the fewest foliar fungicides is the most effective approach for barley disease treatments. However, managing soilborne pathogens in barley plants is problematic due to a deficiency in distinguishing symptoms for diagnosis and the absence of fungicides or nematicides that are effective for these pathogens. Recently, nanotechnology has driven the advancement of creative concepts and agricultural productivity with a broad scope for managing plant infections and pests. The antimicrobial properties of metallic and metal oxide nanoparticulates such as silver, selenium, titanium dioxide, zinc oxide, and iron oxide have been extensively researched. In this chapter, we go over barley disease and the role of nanomaterials in reducing the incidence of disease and diagnosis, as well as barley seed germination, physiology, and nutritional quality of barley grain.KeywordsLeaf rust diseaseNet Blotch diseasePowdery mildewBarley yellow dwarfBarley smutSpot blotchFungicidesNanoparticulate
... DNA biosensors based on nucleic acid recognition have applications such as in electrophoresis analysis of amplified DNA. The applications of DNA-based biosensor analysis extend to the field of food control, process control of raw materials, and traceability in industrial processing plants, and in the field of food control, not only for raw materials but also for process control and traceability in industrial processing plants (Minunni et al. 2005;Mannelli et al. 2003;Bogani et al. 2008). Label-free piezoelectric DNA biosensors present adequate specificity and high sensitivity, allowing rapid and real-time control of DNA hybridisation (Lucarelli et al. 2008;Wu et al. 2007;Sun et al. 2006). ...
Nanotechnology is an emerging technological and scientific breakthrough that can transform agricultural sectors by providing novel tools for the molecular detection of biotic and abiotic stress, and the rapid detection of phytopathogenic diseases. In plants, it has the potential to enhance their capacity to absorb water and nutrients from the soil. Furthermore, nanobiotechnology improves our understanding of crop biology, yields, and nutritional values. The various applications of nanotechnology in agriculture are (1) energy storage, production and conversion (photovoltaic modules); (2) increased agricultural productivity (nanoporous zeolites for prolonged and efficient release of fertilizers); (3) capsules for the specific release of pesticides; (4) the use of biosensors for monitoring the soil quality and plant vitality; (5) pest and phytopathogen detection biosensors; and (6) pesticide biosensors. Nanosensors and intelligent delivery systems based on nano-products are used in the agricultural sector to combat crop pathogens. This nanotechnology seeks to minimize nutrient losses in fertilization and improve crop productivity by optimizing the use of water and nutrients. Nanotechnology provides a wide range of opportunities to produce agro-products based on nanomaterials such as fertilizers, pesticides, herbicides, and nanosensors. These will make it possible to increase the food yield sustainably, reduce the environmental impact and detect infections in plants. This chapter talks about how nanotechnology can be used in plant pathology and how nanomaterials can be used to make biosensors that can detect the main bacterial diseases in maize.KeywordsBiosensorsNanobiotechnologyNanomaterialsNanoparticlesNanosensors
... So, three to four bacterial colonies were dissolved in 500 µL Tris-EDTA (TE) buffer (pH = 8) and killed with incubation at 80 C for 90 min. The thermal attitude (96 C for 8 min, cold shock by ice for 120 s) was applied for directly diagnosis of the target sequence in the non-replicated, and singlestranded of genomic DNA from mentioned strains [49]. In the next step, under optimized conditions, each one of the DNA sequences groups was introduced to the fabricated biosensor surface and the working electrode was rinsed via PBS to eliminate the non-bounded target strands to DNA probe strands. ...
Mycobacterium tuberculosis (M.tb) is a human pathogen due to its slow growth rate and fastidious nature. Tools that can accurately and rapidly track M.tb are vital for the effective treatment of tuberculosis (TB). In present study, a novel genosensor was established for the highly sensitive diagnosis of the specific DNA target inside IS6110 sequence of M.tb genome, using MXene nanosheets and polypyrrole as electrode materials. The multilayer Ti3C2Tx MXene, prepared from the synthesized Ti3AlC2 by delamination process using HCl + LiF solution, presented notable activity in electrocatalysis as an electrode material. Composition information and morphological features of synthesized Ti3C2Tx MXene were studied in detail. Density functional theory (DFT) was applied to explain the structural evolution of Mxene, polypyrrole, nucleotide base, polypyrrole-Mxene hybrid, and nucleotide base-polypyrrole-Mxene intercalation as well as describe their interactions. Under the optimized conditions, the suggested biosensor presented a linear range of 100 fM −25 nM and an excellent coefficient equals 0.9909 and LOD equals 11.24 fM. Experimental results confirmed that this biosensor possessed great sensitivity, stability, and selectivity. This biosensor efficiently determined M.tb in human sputum samples, together with high detection recoveries of 90.52–100.8%.
... 36 The thermal approach (95°C for 6 min, cooling in ice for 90 s) was used for direct detection of the target sequence in the non-amplified genomic DNA of strains. 37 Then, each group of DNA sequences was added to the surface of the modified GCE and the electrode was washed with PBS to remove the unbounded targets to probes. Denaturation of DNA hybrids at the GCE surface has been occurred by immersing it at 0. 5 M NaOH solution (alkaline denaturation method) and aged at room temperature for 6 min. ...
In the perspective of tuberculosis (TB) disease, a necessary issue is the short interval of the correct diagnosis to planning and starting appropriate antibiotic treatment. So, at the first step for the diagnosis of Mycobacterium tuberculosis (M. tb) complex, a fast and reliable technique is necessary. The conventional methods have not the sensitivity, discriminatory power, and enough specificity required for immunocompromised persons. The friendly usage, availability, miniaturization, real-time, and continual monitoring properties of nanobiosensors, an interest attracted to them. The formation of a hybridization reaction in DNA biosensors can provide a possibility for point-of-care infectious detection of M. tb in regions with a high burden of tuberculosis. Here, we have developed a rapid, low-cost, PCR-free with high sensitivity and specificity DNA nanobiosensor for M. tb complex detection, using multi-welled carbon nanotubes, polypyrrole, and potassium-substituted hydroxyapatite (KHAp) nanoparticles. The nanocrystalline powder of KHAp was prepared by a facile alkoxide-based sol-gel method. A selectivity assay using Mycobacterium simiae, Rhodococcus, Nocardia, Corynebacterium, exhibited that the proposed biosensor was specific to M. tb complex. This biosensor showed an appropriate linear relationship (R2 = 0.9906) between the increase in peak current and logarithmic target concentrations from 100 pM to 100 nM, with LOD and LOQ of 50.3 and 167.5 pM, respectively. Its suitable sensitivity was 335.914 μA nM-1cm-2. The response time of this biosensor was 51.3 s. The proposed biosensor remained about 75% of its initial activity after 29 d. The potential application of the nano-biosensor was determined by spike-in experiments to obtain recoveries between 73% and 103.7%. © 2021 The Electrochemical Society ("ECS"). Published on behalf of ECS by IOP Publishing Limited.
... The PCR is a primer-mediated enzymatic amplifi cation of target DNA fragments [1] , whereby the production of target fragments increases at an exponential rate during the reaction. Thus, a trace amount of hereditary material can be amplifi ed millions of times in a few hours for use in other applications, such as DNA sequencing [2] , molecular diagnosis [3,4] , and genetic analysis [5][6][7] . However, PCR introduces biases during amplifi cation; for example, in the aptamer selection process [8][9][10] , during multitemplate amplifi cation [11] , and in the amplifi cation of sequences with extreme base compositions (sequences with mostly G/C or A/T bases) [12][13][14] . ...
Rare earth elements have many uses, and are frequently included in products such as fluorescent materials, hydride batteries, catalytic materials and lasers. In this study, it was observed that trivalent lanthanide ions (Ln[III] ions) appeared to inhibit the synthesis of large fragments in PCR assays, thus resulting in the preferential amplification of shorter sequences. It is therefore speculated that this Ln(III) ion-mediated bias could be utilized to improve the success rates for amplification of shorter products.
... It is challenging to keep the denatured DNA duplex strands apart long time enough for hybridization with the cpDNA. 37 In this study, the 15 nt blocker designed to target a small portion of the tDNA cm sequence was added into the tDNA/tDNA cm solution in the denaturation/renaturation process to hybridize with the tDNA cm , for limiting the rehybridization of tDNA cm with tDNA, and thus releasing the tDNA sequence. To testify the feasibility of the tDNA releasing and detection strategies with our biosensor, the standard target dsDNA (104 bp) of 1 pM was detected with the biosensor rst by using the DPV measurement. ...
Noninvasive diagnosis of Helicobacter pylori (H. pylori) infection is very attractive. This study investigated the single strand DNA (ssDNA) acquisition method from H. pylori in dental plaque, and the integration of our previously developed 43-mer H. pylori DNA biosensor with the obtained target ssDNA (tDNA). Dental plaque samples were collected from 34 patients/volunteers, whose gastric H. pylori infection statuses were tested with the ¹³C urea breath test (UBT). The samples were treated with colony polymerase chain reaction (PCR) to obtain double strand DNA (dsDNA) of 104 basepairs (bp) long. A blocker ssDNA was designed and used in thermal treatment of the dsDNA to release the 104-mer tDNA, which contains the 43-mer DNA sequence in the middle. PCR primers were designed, and the tDNA releasing and detection conditions with the biosensor were optimized. The limit of detection with the biosensor was 12 fM dsDNA. The dental plaque detection results correlated quite well with the UBT results, with a sensitivity of 100%, and specificity of 97%. These results indicate that the residence of H. pylori in dental plaque is highly associated with gastric H. pylori infection, and detection of dental plaque samples with our DNA biosensor is promisingly applicable in noninvasive diagnosis of H. pylori infection.
... Although PCR-free piezoelectric detection of GM DNA from tobacco leaves has been accomplished, this is not the general case for most sensing methods, especially those regarding DNA from foodstuffs. [85] This is a major challenge yet to be conquered in the GMO biosensing field and to achieve this, ultrasensitive devices are required. Methods that combine micro-or nanomaterial-modified surfaces with conductive, area-enhancing materials generally provide the highest sensitivities. ...
In the nearly two decades since genetically modified organisms (GMOs) were first commercialized, genetically engineered crops have gained ground on their conventional counterparts, reaching 185 million hectares worldwide in 2016. The technology has bestowed most of its benefits on enhancing crop productivity with two main traits currently dominating the market: insect-resistant and herbicide-tolerant crops. Despite their rapid and vast adoption by farmers worldwide, GMOs have generated heated debates, especially in European countries (EU), driven mostly by consumers concerned about safety of transgenic foods and about the potential impact on the environment. The need to monitor and to verify the presence and the amount of GMOs in agricultural crops and in food products has generated interest in analytical methods for sensitive, accurate, rapid, and cheap detection of these products. DNA biosensors have been envisioned as a novel DNA-detection technology that would one day substitute current amplification-based methods, providing hand-held, quick, and ultrasensitive gene-level detection. This review summarizes the contributions made in nearly 20 years of research regarding the application of genosensing technology for the qualitative and quantitative determination of transgenic traits.
... These hybridization-based platforms for DNA detection are challenging to deploy in genomic DNA where the efficiency of hybridization is hampered by its large size and complexity. Consequently, the development of an integrated and miniaturized platform for genomic DNA quantification usually requires a combination of a sample pretreatment step, 6,7 uncoiling and cutting the genome, and a sequence-specific detection method. One approach is to integrate a nucleic acid-based sensor and an amplification method, which contributes not only to restricting the size but also to multiplying the target genome. ...
... Therefore, the samples were heated to 95uC in the presence of a large excess (1 mM) of short blocking oligonucleotides ( Table 1). The high temperature ensures efficient thermal denaturation 35 while the presence of blocking oligonucleotides which bind next to probe binding regions guarantees accessibility for probes and prevents re-hybridization during annealing 26,36 , respectively (Fig. 5b). ...
There is an urgent need for rapid and highly sensitive detection of pathogen-derivedDNAin a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp targetDNA fragments of Micrococcus luteus in small sample volumes of 20 mL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of,5 pMand a linear detection range spanning three orders of magnitude.
... Electrochemical aptasensors provide a unique platform for clinical analysis with the advantages of high selectivity, high sensitivity and low cost detections [18][19]. ...
... Nucleic acid detection were approached some years later for Genetically Modified Organism (GMOs) [89,90]. Fragmented PCR amplified samples and directly genomic DNA of transgenic plants (Nicotiana glauca) was studied [91]. ...
Professor Marco Mascini was an internationally acknowledged scientist, teacher of generations of chemists. He passed away on May 12 2015 at the age of 75. During his scientific carrier he made outstanding and innovative contributions to analytical chemistry, especially in the interdisciplinary field of sensors and biosensors. This paper presents an overview of Prof. Mascini's scientific interests and accomplishments in his career that testify the pivotal role of this scientist in the modern analytical chemistry.
... Several methods for studying of DNA hybridization or its interactions with small molecules such as fluorescence, surface Plasmon resonance, quartz crystal microbalance and electrochemistry were used. Among these methods, electrochemical transducers are more attractive for interfacing DNA detection at the molecular level and converting the hybridization event into an analytical signal [2]. In fact high sensitivity, low cost and minimal power requirements of DNA biosensors has caused this device is developed in this field and other fields such as electrochemical clinical diagnosis, environmental monitoring and drug analysis [3]. ...
A new electrochemical DNA hybridization biosensor based on carbon paste electrode (CPE) and ethyl green (EG) is described. The interaction of EG and DNA is investigated in the solution and at the surface of the electrode. The results of electrochemical and spectroscopic studies indicate electrostatic interaction mode between DNA and EG. Hybridization event between the probe and its complementary sequence is investigated by square wave voltammetry of EG accumulated on the CPE. EG displays different signals in the interaction to ssDNA and dsDNA and variation in the EG signal represents the extent of hybridization at the electrode surface. The effects of some experimental variables on the performance of the biosensor are studied and optimized conditions are suggested. The selectivity of the biosensor is studied using some noncomplementary oligonucleotides. Under optimized experimental conditions, limit of detection is calculated 2.0×10-10 M.
... Many electrochemical techniques involving redox molecules, 10 carbon nanotubes, 11,12 and silicon nanowires 13 are novel and successful but lack practicality for clinical applications. Notably, optical techniques may or may not require the use of a label, and label-free methodologies, such as surface plasmon resonance, 14 surface-enhanced Raman scattering, 15 atomic force microscopy, 16 and piezoelectric sensors, 17 have all attracted significant attention. However, the sensitivities of these methods are not comparable to those of label-based methods, and these methods require expensive instrumentation. ...
... Gravity-based DNA sensing instrument includes quartz crystal microbalance (QCM), surface acoustic wave (SAW) and microcantilever (Hansen, Ji et al. 2001;Minunni, Tombelli et al. 2005;Gronewold, Baumgartner et al. 2006). The detection mechanism of QCM and SAW is based on the shift of resonance frequency from quartz crystals or other surface acoustic resonators in response to the change of biomass attached, which finally are interrogated by a piezoelectric signal or a radio frequency signal. ...
... On the other hand, DNA analytes need to be extracted purely from crude samples or non-nucleic acid contaminants before applied to an optical chip, so no obvious photo-induced background from samples can interfere in the final signal. Typical gravity-based DNA sensing instrument includes quartz crystal microbalance (QCM) and microcantilever (Hansen, Ji et al. 2001;Minunni, Tombelli et al. 2005). This type of chips needn't target labeling step for final signal generation and can be re-used multiple times. ...
... Although the above reported examples of probes and probe-surface architectures 820 have been described for oligonucleotide mimicking the DNA target even at very low 821 copy number, or single molecule, only a few works have been published showing 822 actual genomic DNA to demonstrate the use of the techniques for direct detection 823 without amplification. One of the reason for this is the difficulty to target double 824 stranded DNA by displacing one of the two strands, though strategies for overcome 825 this problem have been developed, several examples reported in the text together 826 with further examples are reported in Table 4.1 [153][154][155][156][157][158][159][160][161]for DNA probes and in 827 ...
DNA detection can be achieved using the Watson-Crick base pairing with
oligonucleotides or oligonucleotide analogs, followed by generation of a
physical or chemical signal coupled with a transducer device. The nature
of the probe is an essential feature which determines the performances
of the sensing device. Many synthetic processes are presently available
for "molecular engineering" of DNA probes, enabling label-free and
PCR-free detection to be performed. Furthermore, many DNA analogs with
improved performances are available and are under development; locked
nucleic acids (LNA), peptide nucleic acids (PNA) and their analogs,
morpholino oligonucleotides (MO) and other modified probes have shown
improved properties of affinity and selectivity in target recognition
compared to those of simple DNA probes. The performances of these probes
in sensing devices, and the requirements for detection of unamplified
DNA will be discussed in this chapter. Chemistry and architectures for
conjugation of probes to reporter units, surfaces and nanostructures
will also be discussed. Examples of probes used in ultrasensitive
detection of unamplified DNA are listed.
... 39 Another example shows state of art of QCM sensors for the detection of GMO concentration higher that 0.9% threshold set by the EU. 40 Another mass sensitive QCM-based DNA biosensor for the detection of the hybridization of CaMV 35S promoter sequence (P35S) was developed based on immobilization efficiencies of two procedures. First one, chemisorption of thiolated probe on gold through thiol−gold interaction and blocking thiol procedure ( Figure 9A) and second is the covalent attachment of the amine probe through gluteraldehyde activation of the 13.56 MHz plasma polymerized EDA layer. ...
Tremendous increase has been made in improving the agricultural yield through gene transfer systems of genetically modified organisms (GMOs). However, there are concerns that despite the great progress, introduction of foreign genes into the host genome for new characteristics clearly represents a potential risk for the consumers and environment sustainability. Issues on regulatory approval, safety, and public perception would impact the extent to which GMOs can thrive. With increasing number of new processed foods with novel GMOs advancing into the market, there is an urge to develop a simple to use, sensitive, cost effective, fast and reliable detection device capable of operating on the spot. A vast number of conventional methods are available for detection of GMOs: the protein-based method (enzyme-linked immunosorbent assays (ELISA)) or DNA based such as polymerase chain reaction (PCR) and microarray method (known as DNA chips or genosensor). However, very few have been suitable for real-time and reliable DNA analysis. Biosensors are cutting-edge analytical tools that have a great promise and potential in detecting GMOs in wide range of food products, from maize flour to a cookie. The focus of this review is to discuss the potential of DNA biosensors (genosensors) for GMO identification/detection based on optical, piezoelectric and electrochemical transducers reported from the year 2002 to date. Through this review we anticipate to envision strong scientific contributions and public awareness towards making adequate food decisions.
Biosensors are target-specific affinity based analytical tools. Their applications are vast in areas such as clinical diagnosis, food quality, environment monitoring and in other fields as well where timely and reliable analysis are required. Biosensing technology is gradually developing to produce nove.l biosensors for point-of-care diagnosis. The involvement of new advancements such as aptamer-based technologies and nano-bioelectronic technologies have great potentialto lead to the development of next generation biosensors with improved sensitivity, specificity, portability and cost effectiveness. This review emphasizes several aspects of biosensors such as their classification, mechanism involved and their biomedical applications.
Constant efforts have been devoted to exploring new disinfection byproducts in drinking water causally related to adverse health outcomes. In this study, five halogenated nucleobases were identified as emerging disinfection byproducts in drinking water, including 5-chlorouracil, 6-chlorouracil, 2-chloroadenine, 6-chloroguanine, and 5-bromouracil. We developed a solid phase extraction-ultraperformance liquid chromatography-tandem mass spectrometry method with the limits of detection (LOD) and recoveries ranging between 0.04-0.86 ng/L and 54-93%, respectively. The detection frequency of the five halogenated nucleobases ranged from 73 to 100% with a maximum concentration of up to 65.3 ng/L in the representative drinking water samples. The cytotoxicity of the five identified halogenated nucleobases in Chinese hamster ovary (CHO-K1) cells varied with great disparity, in which the cytotoxicity of 2-chloroadenine (IC50 = 9.4 μM) is appropriately three times higher than emerging DBP 2,6-dichloro-1,4-benzoquinone (IC50 = 42.4 μM), indicating the significant toxicological risk of halogenated nucleobase-DBPs. To the best of our knowledge, this study reports the analytical method, occurrence, and toxicity of halogenated nucleobase-DBPs for the first time. These findings will provide a theoretical basis for further research on probing the relationship between its mutagenicity and human health risk.
In this review, we provide an overview of the nanopaper biosensors used for point of care (POC) diagnostics in various fields such as biomedical, environmental, food safety, and agriculture. The lateral flow assays (LFAs) comprising nanoparticles have drawn the interest of researchers due to their high sensitivity, low cost, lower time consumption, lack of equipment, and easy handling characteristics. These assays have become an integral part of the health sector all over the world over a short time period and are extensively being engaged in diagnosis of viral infections in regions low on resources. A large number of innovative approaches have been introduced in making these test-strips or nanopaper biosensors as quickly as possible, because of their ease of operation, cheaper cost, and quick results, even with the simplest setups or in pathological laboratories, etc. Keeping all this in mind, we have reviewed the nanoparticles based lateral flow test strips (LFTS), their composition, working methods, reaction mechanisms, detection methods, and applications in different fields. We also provide an overview of paper-based microfluidic analytical devices (μPADs) and our perspective about the future trends which may facilitate understanding their applications.
Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria.
For on-site molecular diagnostics, a pre-treatment step for isolation of nucleic acid from clinical samples on site is desired. However, conventional commercialized silica-based nucleic acid isolation kits require repetitive pipetting and a centrifugation or permanent magnet for buffer exchange. In this study, we developed a poly(3,4-dihydroxy-L-phenylalanine) (L-DOPA)-coated swab that can absorb and desorb DNA depending on pH of buffers and a portable integrated DNA isolation device that comprises integrated chambers containing DNA isolation buffers. The poly(L-DOPA)-coated swab interacts with each buffer by passing through the membrane between the integrated chambers. Our device involves a simple operation and does not require any large equipment or skilled experts. By connecting the device with an automated polymerase chain reaction system, an isothermal amplification system, or a non-amplified DNA detection method, on-site molecular diagnosis of various diseases can be quickly realized.
A polymerization reaction was employed as a signal amplification method to realize direct visualization of gender-specific DNA extracted from human blood in a polymerase chain reaction (PCR)-free fashion. Clear distinction between X and Y chromosomes was observed by naked eyes for detector-free sensing purpose. The grown polymer films atop X and Y chromosomes were quantitatively measured by ellipsometry for thickness readings. Detection assays have been optimized for genomic DNA recognition to a maximum extent by varying the selection of the proper blocking reagents, the annealing temperature, and the annealing time. Traditional PCR and gel electrophoresis for amplicon identification were conducted in parallel for performance comparison. In the blind test for blood samples examined by the new approach, 25 out of 26 were correct and one was false negative, which was comparable to, if not better than, the PCR results. This is the first time our Amplification-by-Polymerization technique being used for chromosome DNA analysis. The potential of adopting the described sensing technique without PCR was demonstrated, which could further promote the development of a portable, PCR-free DNA sensing device for point-of-need applications.
An on-surface isothermal helicase-dependent amplification is devised for simple, point-of-need quantification of bacterial genome. The method relies on the enzyme-extension of a thiol-modified reverse primer anchored to indium tin oxide...
Biosensors and related bioanalytical tools are, nowadays, full-accepted systems for many applications in modern analytical chemistry. A biosensor is defined by International Union of Pure and Applied Chemistry as a self-contained integrated device that is capable of providing specific quantitative or semiquantitative analytical information using a biological recognition element (biochemical receptor), which is retained in direct spatial contact with a transduction element. A biosensor should be clearly distinguished from a bioanalytical system, which requires additional processing steps. The peculiar characteristics of biosensors allow them to complement current screening and monitoring methods, especially when continuous, real-time, in situ analysis is required. In this chapter, the basic rules for the development of biosensors are explained. The different bioreceptors are briefly described as well as the different transduction techniques. The critical evaluation of advantages and drawbacks of these analytical tools are included. New trends in the design of biosensors are also discussed.
Nickel doped zinc oxide (Ni-ZnO) thin film based matrix is used for fabricating highly stable and sensitive DNA biosensor for detecting a life-threatening disease, Meningitis. The present DNA biosensor has been fabricated after the immobilization of 23mer oligonucleotide sequence of DNA over the surface of Ni-ZnO/ITO electrode via. electrostatic interaction. Scanning electron microscopy (SEM) studies show the formation of nanostructured Ni doped ZnO thin film surface morphology which facilitates higher loading of single stranded thiolated DNA (ss th-DNA) molecules over the Ni doped ZnO matrix. The ss th-DNA/Ni-ZnO/ITO bioelectrode response studies were done using differential pulsed voltammetry (DPV) in the methylene blue (MB) mediated buffer. Linear response over wide DNA concentration (5 ng/μl–200 ng/μl) was obtained with a high sensitivity of 49.95 μA/decade. The present biosensor is found to exhibit very low detection limit (5 ng/μl) with a quick hybridization time of only 30 s. Electrochemical impedance spectroscopy (EIS) has also been used for studying the sensing response of ss th-DNA/Ni-ZnO/ITO bioelectrode.
The property of piezoelectric crystals of vibrating under the influence of an electric field, and the relationship between the resonant frequency changes and the mass of molecules adsorbed or desorbed from the surface of the crystal are the basis of the transduction mechanism in piezoelectric biosensors. This chapter proposes an overview of literature in the last three years on this subject, with focus on medical applications classifying the different piezoelectric biosensors depending on the biomolecule coupled to the piezoelectric sensor (antibodies, nucleic acids and bio-mimetic receptor, mainly aptamers).
As an important member of the tool enzymes, exonuclease is a kind of hydrolytic enzymes without strict base sequence dependence. In recent years, by taking advantage of different hydrolysis ways of exonuclease and nanotechnology, cycle effect of enzyme digestion, aptamer, non Watson-Crick base pairing system by metal ions, fluorescent nucleic acid probes, electrochemical methods etc., a series of exonuclease-assisted signal amplification strategies have been developed, which have played a key role in improving the sensitivity of detection method. Therefore, exonucleases have been widely used in highly sensitive detection of nucleic acids, proteins, ions, small molecules and so on. To understand it better and apply it well in the future, the application progresses of exonuclease-assisted signal amplification strategies in biochemical analysis have been summarized in this review.
Tuberculosis (TB) is a deadly infectious disease caused by Mycobacterium tuberculosis (MTB) that particularly attacks the lungs. According to the World Health Organization (WHO) report of 2012, approximately 8.6 million people developed TB globally, approximately 1.3 million of those people died, and India contributes approximately 26% of the global TB burden. Vaccines like Bacillus Calmette-Guerin (BCG) and various drugs including isoniazid, rifampicin, and their derivatives are available for the management of TB; unfortunately, none is 100% effective. Because of the continuous and excessive use of these drugs, the bacterium has developed resistance. Therefore, novel technology-based methods for the diagnosis and treatment of TB to help reduce the global burden of TB are needed.This chapter focuses on the use of different nanotechnological methods, including nano-based drug delivery using different nanomaterials and use of nanobiosensors, for tackling the problem related to TB. It is also focuses on different topics such as distribution of TB, disease severity, available treatment, multidrug resistance, and limitation of chemotherapy.
The most suitable approach as an analytical alternative methodology for the detection of trace clinical analytes is based on affinity sensing, in which the immobilized biological element (receptor) can be an antibody, a receptor (natural or synthetic), a nucleic acid, or an aptamer.
Immunosensors, based on antibody–antigen interactions, and DNA‐based sensing, based on nucleic acid hybridization, are first reported. Finally an aptasensor, based on a new category of relevant receptors, nucleic acid sequences selected in vitro named aptamers, is shown. The reported examples deal with two different transduction principles, electrochemical and piezoelectric, applied to analyte detection of clinical interest. The development of immunosensors for progesterone detection by electrochemical sensing is first reported. Then DNA‐based sensing, for point mutation detection in the b‐globin gene, occurring in b‐thalassemia, is shown. Finally aptasensing for thrombin detection is discussed.
Both single‐use and multiuse sensing have been shown to be suitable for trace analysis in clinical chemistry. Both approaches possess sensitivity, reproducibility, and analysis times compatible with the final application.
A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65 °C, providing 3×106 amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4 h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available.
A novel electrogenerated chemiluminescence (ECL) aptasensor for ultrasensitive detection of thrombin incorporating an auxiliary probe was designed by employing specific anti-thrombin aptamer as a capture probe and a ruthenium(II) complex-tagged reporter probe as an ECL probe and an auxiliary probe to assist the ECL probe close to the surface of the electrode. The ECL aptasensor was fabricated by self-assembling a thiolated capture probe on the surface of gold electrode and then hybridizing the ECL probe with the capture probe, and further self-assembling the auxiliary probe. When analyte thrombin was bound with the capture probe, the part of the dehybridized ECL probe was hybridized with the neighboring auxiliary probe, led to the tagged ruthenium(II) complex close to the electrode surface, resulted in great increase in the ECL intensity. The results showed that the increased ECL intensity was directly related to the logarithm of thrombin concentrations in the range from 5.0×10(-15)M to 5.0×10(-12)M with a detection limit of 2.0×10(-15)M. This work demonstrates that employing an auxiliary probe which exists nearby the capture probe can enhance the sensitivity of the ECL aptasensor. This promising strategy will be extended to the design of other biosensors for detection of other proteins and genes.
Functionalized microspheres are frequently used in the detection of biomolecules. A fundamental understanding of the mechanisms involved in enzymatic assays is required to estimate a method's utility. The mathematical methodology is illustrated through a theoretical analysis to assess the performance of a diagnostic method based on the horseradish peroxidase catalyzed reaction to detect pathogens in clinical specimens. The first part of the analysis focuses on the collection of target DNA molecules onto a functionalized fiber in a lysis micro-reactor (LMR). Expressions are derived for hybridization rates that include convective transport. In the next step the fiber is contacted with functionalized chitosan microspheres containing horse radish peroxidase. Chitosan microspheres are conjugated to the fiber with the target DNA acting as a unique tether. The final step is the release of chitosan microspheres in a chromogenic substrate and monitoring absorption changes. The analysis provides estimates of the method sensitivity and processing time and we show that DNA with a concentration as low as 10−4 copies per milliliter can be detected in less than 30 minutes. The modeling methodology presented can readily be extended to similar enzymatic, microsphere-based assays for quantitative purposes and feasibility studies.
In the last 2 decades, Surface Plasmon Resonance (SPR) sensing has played a relevant role in many biochemical and biotechnological fields, and various kinds of biorecognition elements are currently employed in a wide range of fields such as food processing, environmental monitoring and clinical/diagnostics analyses. At present, SPR-based sensing represents one of the main optical biosensor technologies. The most attractive and powerful advancement of SPR-based optical detection is the SPR imaging (SPRi) technique (also termed "SPR microscopy"), which couples the sensitivity of scanning angle SPR measurements with the spatial capabilities of imaging. In recent years SPRi has gained attention mainly in affinity-biosensors research field and actually represents a promising sensing platform for the probing of biomolecules in array format, showing to be a highly versatile system. In this work we will report about the development of a multiarray SPRi affinity sensor. In particular the system has been studied using antibody-antigen interactions food and/or clinical controls. The analytical parameters of the systems will be discussed.
Piezoelectric sensing has been widely applied for affinity sensing, and
recently sensitive DNA detection has been reported in different matrices
for different analytes (i.e. target sequences). In this chapter, the
detection principle and the approaches used in DNA-based sensing with
focus on detection of microsatellite DNA, present in high number of copy
as well as target sequence detection of genes present in one or few copy
number per haploid genome will be presented and discussed. Particular
attention will be devoted to the pre-analytical steps which may
influence the sensor response to the target analyte such as genomic DNA
fragmentation and denaturation. Comparison between immobilization
chemistries is also presented. In particular, finding in microsatellite
detection with both biotinylated and thiolated probes is reported and
discussed.
Food safety and quality are very important issues receiving a lot of
attention in most countries by producers, consumers and regulatory and
control authorities. In particular, DNA analysis in food is becoming
popular not only in relation to genetically modified products (GMOs), in
which DNA modification is the "clue" of the novelty, but also in other
fields like microbiology and pathogen detection, which require long
times for the cultivation and specially in cases in which the
microorganisms are not cultivable like some viruses, as well as for
authenticity and allergen detection. A new topic concerning
"nutrigenetics and nutrigenomics" has also been mentioned, very
important but still in its infancy, which could lead in the future to a
personalized diet. In this chapter we have described the main areas of
food research and fields of application where DNA analysis is being
performed and the relative methods of detection, which are generally
based on PCR. The possibility/opportunity to detect DNA without previous
amplification (PCR-free) will be discussed. We have examined the
following areas: (1) genetically modified foods (GMOs); (2) food
allergens; (3) microbiological contaminations; (4) food authenticity;
(5) nutrigenetics/nutrigenomics.
Genetic analyses performed in health laboratories involve adult
patients, newborns, embryos/fetuses, pre-implanted pre-embryos,
pre-fertilized oocytes and should meet the major medical needs of
hospitals and pharmaceutical companies. Recent data support the concept
that, in addition to diagnosis and prognosis, genetic analyses might
lead to development of personalized therapy. Novel frontiers in genetic
testing involve the development of single cell analyses and non-invasive
assays, including those able to predict outcome of cancer pathologies by
looking at circulating tumor cells, DNA, mRNA and microRNAs. In this
respect, PCR-free diagnostics appears to be one of the most interesting
and appealing approaches.
In this work, we demonstrated a novel sensitive sandwich-type pseudobienzyme aptasensor for thrombin detection. Greatly amplified sensitivity was based on mesoporous silica-multiwalled carbon nanotube (mSiO2@MWCNT) nanocomposites as enhanced materials and a pseudobienzyme electrocatalytic system. Firstly, the mSiO2@MWCNT nanocomposites not only have good biocompatibility and a suitable microenvironment for stabilizing the aptamer assembly, but also can load large amounts of electron mediator thionine (Thi), platinum nanoparticles (PtNPs) and hemin/G-quadruplex bioelectrocatalytic complex. Moreover, in the presence of H2O2 in an electrolytic cell, the synergistic reaction of PtNPs and hemin/G-quadruplex bioelectrocatalyzed the reduction of H2O2, dramatically amplifying the response signals of electron mediator Thi and improving the sensitivity. Secondly, dendrimer functionalized reduced graphene oxide (PAMAM-rGO) as the biosensor platform enhanced the surface area for the immobilization of abundant primary aptamers as well as facilitated electron transfer from Thi to the electrode, thus amplifying the detection response. Using the above multiple effects, the approach showed a high sensitivity and a wider linearity for the detection of thrombin in the range between 0.0001 nM and 80 nM with a detection limit of 50 fM. This new design avoided the fussy labeling process and the spatial distribution of each sequentially acting enzyme, which provided an ideal candidate for the development of a sensitive and simple bioanalytical platform.
Hybridization-based assays for DNA detection often use single-stranded DNA (ssDNA) probes to capture ssDNA targets in solution. Unfortunately, these assays are often not able to detect double-stranded DNA (dsDNA). Here, we achieve highly sensitive dsDNA target detection by including short oligonucleotide sequences during denaturing and cooling. After performing an isothermal nucleic acid amplification technique (Rolling Circle Amplification, RCA), these captured dsDNA targets are labeled, allowing single amplified molecules to be imaged and counted. This detection method was first applied to the detection of PCR-generated (polymerase chain reaction) dsDNA targets, yielding a limit of detection of 4.25 fM. As an application of the developed assay, the detection of extracted Mycobacterium tuberculosis (M. tb.) genomic DNA was attempted. A M. tb.-specific target was detected with high specificity compared to similar bacteria, and a detection limit of 10000 colony forming units (cfu) ml−1 was achieved, close to the sensitivity required for clinical diagnosis.
A biosensor approach using optical transduction based on surface plasmon resonance is adopted for the direct detection of target sequences in genomic DNA of several origins. In particular, the sensor was applied to three different sequences, representative of different living organisms from plant to human and differing in terms of copies in the relative genome.The sample pretreatment consisted in a fragmentation with restriction enzymes, followed by an optimized denaturation step. The biosensor resulted specific both with oligonucleotides and with genomic non‐amplified DNA. The sensor could represent an alternative method to traditional biomolecular techniques for the identification of DNA sequences in digested DNA, bypassing the amplification step.
Polymerase chain reaction (PCR) is one of the most important technologies of modern molecular biology; there are still many problems in PCR that cannot be overcome. It is significant to study the ways of optimizing the PCR amplification. Owing to several limitations in conventional ways, PCR amplification still turns out not so satisfactory. With the development of nanotechnology, the advantages of nanomaterials have attracted more attention. The superficial and dimensional properties of nanomaterials have special effects on the structure and function of biological macromolecules such as proteins and nucleic acids. Therefore, it possesses very important academic and applied meanings to study the ways and technologies of optimizing PCR based on nanomaterials. In this review, the recent researches of nanomaterials based on PCR, with 41 references, are summarized, particularly the developing trends and prospects in the future.
A family of new imidazolium salts derived from natural amino acids has been synthesized and tested for NMR enantiodiscrimination, as chiral shift reagents, of carboxylic acids. These imidazolium receptors contain different structural modifications and the splitting of the signals of the acids, after addition of the corresponding CSRs, depends on these structural variables. Compound 8b exhibited the strongest chiral solvating properties for racemic Mosher acid and was recognized as a suitable CSR for the determination of its enantiomeric composition.
The sensitive and rapid detection of pathogenic DNA is of tremendous importance in the field of diagnostics. We demonstrate the ability of detecting and quantifying single- and double-stranded pathogenic DNA with picomolar sensitivity in a bead-based fluorescence assay. Selecting appropriate capturing and detection sequences enables rapid (2 h) and reliable DNA quantification. We show that synthetic sequences of S. pneumoniae and M. luteus can be quantified in very small sample volumes (20 muL) across a linear detection range over four orders of magnitude from 1 nM to 1 pM, using a miniaturized wide-field fluorescence microscope without amplification steps. The method offers single molecule detection sensitivity without using complex setups and thus volunteers as simple, robust, and reliable method for the sensitive detection of DNA and RNA sequences.
We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.
A DNA-based sensor for the label-free, real-time detection of highly repeated sequences in non-amplified DNA is reported. The transduction principle is a piezoelectric quartz crystal and the immobilisation chemistry is based on the direct coupling of thiol-modified probes on the gold electrode surface. The sequence chosen as probe is internal to a region of the satellite 13 DNA from Bos taurus.The sensor was developed using bovine DNA commercially available, alone or mixed at different percentages with DNA of a different origin (porcine DNA: Sus scrofa). Real samples (DNA extracted from bovine animal muscle) have also been tested.The sample pre-treatment consisted only in a fragmentation with restriction enzymes, followed by an optimised denaturation step. No amplification by polymerase chain reaction was carried out.The developed biosensor resulted very specific reproducible (CV% of 11%) both with synthetic oligonucleotides and with genomic non-amplified DNA. In both cases, the tested negative controls did not give significant frequency shifts. Moreover, the signals obtained with samples containing bovine DNA, alone or mixed with DNA of a different origin, are comparable. The sensor could represent a method alternative to traditional biomolecular techniques, for the identification of species-specific DNA sequences, directly in enzymatically digested DNA, bypassing the amplification step.
A piezoelectric affinity sensor, based on DNA hybridisation has been studied for applications to Genetically Modified Organisms (GMOs) detection. The thiol/dextran modified surfaces were coupled to streptavidin for immobilising 5'-biotinyltead probes (25-mer). The probes sequences were respectively internal to the amplified product of P35S and T-NOS. These target sequences were chosen on the base of their wide presence in GMOs. The system has been optimised using synthetic complementary oligonucleotides (25-mer) and the specificity of the system tested with a non-complementary oligonucleotide (23-mer). The hybridisation study was performed also with samples of DNA isolated from CRM (Certified Reference Materials) soybean powder containing 2% of transgenic material and amplified by PCR. Non amplified genomic or plasmidic DNA was also used. The developed system was very specific, binding only the complementary DNA strand. The CV% was 20% both with synthetic oligonucleotides and PCR amplified samples. The sensor signal was independent of the sample dilution but the system is still at a semi-quantitative level.
Wide-scale DNA testing requires the development of small, fast and easy-to-use devices. This article describes the preparation,
operation and applications of biosensors and gene chips, which provide fast, sensitive and selective detection of DNA hybridization.
Various new strategies for DNA biosensors and gene chips are examined, along with recent trends and future directions. The
integration of hybridization detection schemes with the sample preparation process in a ‘Lab-on-a-Chip’ format is also covered.
While the use of DNA biosensors and gene chips is at an early stage, such devices are expected to have an enormous effect
on future DNA diagnostics.
Electrochemists are developing fast and easy methods for determining nucleic acid sequences and DNA damage.
Replication, precipitation, and amplification: Polymerase or reverse transcriptase induced replication of DNA/RNA on a transducer (electrode or piezoelectric crystal) leads to the ultrasensitive specific electronic transduction of viral genomes. Biotin tags (B) on the double-stranded assembly provide docking sites for a conjugate between avidin (A) and an alkaline phosphatase (AP). Enzyme biocatalysis of substrate (S) to the insoluble product (P), which precipitates onto the transducer (yellow surface), provides amplification in the analysis of the target DNA.
Fiber optic biosensors operated in a total internal reflection format were prepared based on covalent immobilization of 25mer lacZ single-stranded nucleic acid probe. Genomic DNA from Escherichia coli was extracted and then sheared by sonication to prepare fragments of approximately 300mer length. Other targets included a 25mer fully complementary lacZ sequence, 100mer polymerase chain reaction (PCR) products containing the lacZ sequence at various locations, and non-complementary DNA including genomic samples from salmon sperm. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on extent or speed of hybridization of complementary oligonucleotides. Detection of genomic fragments containing the lacZ sequence was possible in as little as 20 s by observation of the steady-state fluorescence intensity increase or by time-dependent rate of fluorescence intensity changes.
This study presents a microfabricated device for polymerase chain reaction (PCR) with a flow-through manner for use in environmental microbiology. The device was developed utilizing photolithography and softlithography techniques and evaluated as one component of a totally integrated in situ gene analysis system. The developed device was composed of a glass-based “temperature control chip” and polydimethylsiloxane (PDMS)-based “microchannel chip”. For the temperature control chip, six heaters made from indium tin oxide (ITO) are placed on a glass substrate to define three uniform temperature zones for flow-through PCR. On the each heater, a platinum (Pt) line was placed as a temperature sensor. The PDMS microchannel structure was fabricated by using a molding method with a negatively patterned mold master. The width and depth of folded microchannel was 100 μm and total length for 30 cycles of flow-through PCR was approximately 3.0 m. With the flow-through PCR device, 580 and 1450 bp (base pairs) of DNA fragments were successfully amplified from Escherichia coli genomic DNA and directly from untreated cells.
Hybridization rates of sheared, genomic E. coli DNA in 0.14 M, pH 6.7 phosphate buffer at 65 degrees C were determined by: (1) observing the rate of absorbance decrease at 260 nm due to self-hybridization in solution; and (2) measurement of the rate of mass increase caused by hybridization between DNA in solution and DNA photografted to polystyrene. The latter measurement was done using a quartz crystal microbalance (QCM). In both the spectrophotometric and QCM experiments the probe was identical to the target, as both were taken from the same sample of sheared E. coli DNA. In the QCM measurements, viscoelastic effects were made negligible by drying the biopolymer layer on the QCM's surface before taking the frequency readings. Our purpose was to explore the effect of immobilizing DNA on its hybridization rate constant. A second-order constant of 2.32 +/- 0.09 x 10(-6) ml microg(-1) s(-1), n = 14, for hybridization in solution was obtained spectrophotometrically, while the QCM experiment gave a constant of 2.2 +/- 0.3 x 10(-6) ml microg(-1) s(-1), n = 6. These values are not statistically different. The reaction half-lives for the spectrophotometric and QCM experiments were 6.5 h and 13 min, respectively. The shorter half-life on the QCM can be explained solely by the much greater reactant concentration in the QCM experiment. About 25% of the DNA was inactivated by the attachment reaction. After correcting for this, the surface-attached DNA hybridized with the same rate constant as DNA free in solution. Therefore, it is concluded that, in these specific experiments with genomic DNA, the immobilized regions must have been short compared to the length of the molecules. The data demonstrate the high hybridization rate obtainable when nucleic acids are hybridized in a thin-film, micro-volume reaction on a non-porous surface.
Biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect genetically modified Roundup Ready soybean gene sequences. We first immobilized, on SA sensor chips, single-stranded biotinylated oligonucleotides containing soybean lectin and Roundup Ready gene sequences, and the efficiency of hybridization to oligonucleotide probes differing in length was determined. Second, we immobilized biotinylated PCR products from nontransgenic soybeans (genomes carrying only the lectin gene), as well as from genetically modified Roundup Ready soybean, and we injected the oligonucleotide probes. Furthermore, we used the sensor chips carrying either lectin and Roundup Ready soybean PCR products or 21-mer oligonucleotide as probes, and we injected both nonpurified and purified asymmetric PCR products. The results obtained show that 13 and 15 mer oligonucleotides are suitable probes to detect genetically modified Roundup Ready soybean gene sequences (either target oligonucleotides or PCR products) under standard BIA experimental conditions. By contrast, when 11 mer DNA probes were employed, no efficient hybridization was obtained. All the SPR-based formats were found to be useful for detection of Roundup Ready gene sequences, suggesting that these procedures are useful for the real-time monitoring of hybridization between target single-stranded PCR products, obtained by using as substrates DNA isolated from normal or transgenic soybeans, and oligonucleotide or PCR-generated probes, therefore enabling a one-step, nonradioactive protocol to perform detection.
Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.
A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.
The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.
A novel surface enzymatic reaction scheme that amplifies the optical response of RNA microarrays to the binding of complementary DNA is developed for the direct detection and analysis of genomic DNA. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from DNA-RNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance (SPR) imaging, DNA oligonucleotides can be detected at a concentration of 1 fM. This enzymatically amplified SPR imaging methodology is then utilized to detect and identify the presence of the TSPY gene in human genomic DNA without PCR amplification.
With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) combined with hybridization technique was applied to detect the GMOs in genetically modified (GM) soybeans and papayas for the first time. Whether the soybeans and the papayas contain GM components was discriminated by detecting the Cauliflower mosaic virus 35S (CaMV35S) promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM soybeans and papayas. The technique may provide a new means in GMOs detection due to its simplicity and high efficiency.
This paper describes the optimisation and the analytical performances of an enzyme-based electrochemical genosensor, developed using disposable oligonucleotide-modified screen-printed gold electrodes. The immobilisation of a thiol-tethered probe was qualitatively investigated by means of faradic impedance spectroscopy. Impedance spectra confirmed that the thiol moiety unambiguously drives the immobilisation of the oligonucleotide probe. Furthermore, both probe surface densities and hybridisation efficiencies were quantified through chronocoulometric measurements. Electrochemical transduction of the hybridisation process was also performed by means of faradic impedance spectroscopy, after coupling of a streptavidin-alkaline phosphatase conjugate and bio-catalysed precipitation of an insoluble and insulating product onto the sensing interface. Chronocoulometric results allowed discussion of the magnitude of hybridisation signals in terms of probe surface densities and their corresponding hybridisation efficiency. The genosensor response varied linearly (r2 = 0.9998) with the oligonucleotide target concentration over three orders of magnitude, between 12 pmol/L and 12 nmol/L. The estimated detection limit was 1.2 pmol/L (i.e., 7.2 x 10(6) target molecules in 10 microL of sample solution). The analytical usefulness of the impedimetric genosensor was finally demonstrated analysing amplified samples obtained from the pBI121 plasmid and soy and maize powders containing 1 and 5% of genetically modified product. Sensing of such unmodified amplicons was achieved via sandwich hybridisation with a biotinylated signaling probe. The electrochemical enzyme-amplified assay allowed unambiguous identification of all genetically modified samples, while no significant non-specific signal was detected in the case of all negative controls.
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