Molecular Rapid Test for Identification of Tuna Species.

Tuna is an excellent food product, relatively low in calories, that is recommended for a balanced diet. The continuously increasing demand, especially for bluefin-tuna-based food preparations, and its relatively high market price make adulteration by intentionally mixing with other lower-priced tunas more prospective. The development of rapid methods to detect tuna adulteration is a great challenge in food analytical science. We have thus developed a simple, fast, and low-cost molecular rapid test for the visual detection of tuna adulteration. It is the first sensor developed for tuna authenticity testing. The three species studied were Thunnus thynnus (BFT), Thunnus albacares, and Katsuwonus pelamis. DNA was isolated from fresh and heat-treated cooked fish samples followed by PCR. The PCR products were hybridized (10 min) to specific probes and applied to the rapid sensing device. The signal was observed visually in 10-15 min using gold nanoparticle reporters. The method was evaluated employing binary mixtures of PCR products from fresh tissues and mixtures of DNA isolates from heat-treated tissues (canned products) at adulteration percentages of 1-100%. The results showed that the method was reproducible and specific for each tuna species. As low as 1% of tuna adulteration was detected with the naked eye.

Fish DNA Sensors for Authenticity Assessment-Application to Sardine Species Identification.

Food and fish adulteration is a major public concern worldwide. Apart from economic fraud, health issues are in the forefront mainly due to severe allergies. Sardines are one of the most vulnerable-to-adulteration fish species due to their high nutritional value. Adulteration comprises the substitution of one fish species with similar species of lower nutritional value and lower cost. The detection of adulteration, especially in processed fish products, is very challenging because the morphological characteristics of the tissues change, making identification by the naked eye very difficult. Therefore, new analytical methods and (bio)sensors that provide fast analysis with high specificity, especially between closely related fish species, are in high demand. DNA-based methods are considered as important analytical tools for food adulteration detection. In this context, we report the first DNA sensors for sardine species identification. The sensing principle involves species recognition, via short hybridization of PCR-amplified sequences with specific probes, capture in the test zone of the sensor, and detection by the naked eye using gold nanoparticles as reporters; thus, avoiding the need for expensive instruments. As low as 5% adulteration of Sardina pilchardus with Sardinella aurita was detected with high reproducibility in the processed mixtures simulating canned fish products.

Screening Method for the Visual Discrimination of Olive Oil from Other Vegetable Oils by a Multispecies DNA Sensor.

Olive oil is a prominent agricultural product which, in addition to its nutritional value and unique organoleptic characteristics, offers a variety of health benefits protecting against cardiovascular disease, cancer, and neurodegenerative diseases. The assessment of olive oil authenticity is an extremely important and challenging process aimed at protecting consumers and producers. The most frequent adulteration involves blending with less expensive and readily available vegetable/seed oils. The methods for adulteration detection, whether based on changes in metabolite profiles or based on DNA markers, require advanced and expensive instrumentation combined with powerful chemometric and statistical tools. To this end, we present a simple, multiplex, and inexpensive screening method based on the development of a multispecies DNA sensor for sample interrogation with the naked eye. It is the first report of a DNA sensor for olive oil adulteration detection with other plant oils. The sensor meets the 2-fold challenge of adulteration detection, i.e., determining whether the olive oil sample is adulterated and identifying the added vegetable oil. We have identified unique, nucleotide variations, which enable the discrimination of seven plant species (olive, corn, sesame, soy, sunflower, almond, and hazelnut). Following a single PCR step, a 20 min multiplex plant-discrimination reaction is performed, and the products are applied directly to the sensing device. The plant species are visualized as red spots using functionalized gold nanoparticles as reporters. The spot position reveals the identity of the plant species. As low as <5-10% of adulterant was detected with particularly good reproducibility and specificity.

Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays.

The COVID-19 pandemic proved the great usefulness of lateral flow tests as self- and rapid tests. The rapid expansion of this field requires the design and validation of novel, affordable, and versatile technologies for the easy fabrication of a variety of lateral flow devices. In the present work, we have developed a new, simple, and cost-effective system for the dispensing of reagents on the membranes of lateral flow devices to be used for research purposes. The 3D printing technology is integrated, for the first time, with simple and inexpensive tools such as a technical pen and disposable pipet tips for the construction of the test and the control areas of the devices. We also used this system for the automated fabrication of spots on the membrane for multiplex analysis. The devices were applied for the detection of proteins/antibodies and single- and double-stranded DNA targets. Also, devices with multiple biosensing areas on the membrane were constructed for the simultaneous detection of different analytes. The proposed system is very simple, automated, and inexpensive and has provided rapid and reproducible construction of lateral flow devices. Compared to a commercially available automated dispenser, the devices showed similar detection capabilities and reproducibility in various real samples. Moreover, contrary to the existing dispensers, the proposed system does not require any gas or costly precision pumps and syringes for the deposition. In conclusion, the developed 3D printer-based system could be an extremely useful alternative for research laboratories for the construction of lateral flow devices of various assay configurations.

Multifold improvement in allergen detection capability of dipstick-type immunosensors via macromolecular crowding.

Dipstick-type lateral flow immunosensors are used widely for on-site detection of food allergens. The weakness of the immunosensors of this type, however, is their low sensitivity. Contrary to current methods, that focus on improving detection capability through the introduction of novel labels or multistep protocols, this work exploits macromolecular crowding to modify and regulate the microenvironment of the immunoassay, thus promoting the interactions that are responsible for allergen recognition and signal generation. The effect of 14 macromolecular crowding agents was explored using, as a model, commercially available and widely applied dipstick immunosensors, which are already optimized in terms of reagents and conditions for peanut allergen detection. An about 10-fold improvement in detection capability was achieved by using polyvinylpyrrolidone, Mr 29,000, as a macromolecular crowder without compromising simplicity and practicality. The proposed approach is complementary to other methods of improving the sensitivity by using novel labels. Because biomacromolecular interactions have a fundamental role in all types of biosensors, we foresee that the proposed strategy will also find applications in other biosensors and analytical devices.

A universal lateral flow assay for microRNA visual detection in urine samples.

MicroRNAs (miRNAs) have been emerged as novel and significant biomarkers in liquid biopsy that can be found in different body fluids. Several techniques have been developed and applied for miRNAs analysis, including nucleic acid-based amplification methods, next generation sequencing, DNA microarrays and new genome-editing methods. These methods, however, are time-consuming and require expensive instruments and specially trained personnel. Biosensors, on the other hand, are alternative and valuable analytical/diagnostic tools due to their simplicity, cost-effectiveness, rapid analysis and ease of use. Several biosensors, especially nanotechnology-based ones, have been developed for miRNA analysis that are based either on target amplification or signal amplification and target re-cycling for sensitive detection. At this point of view, we have introduced a new and universal lateral flow assay in combination with reverse transcription - polymerase chain reaction (RT-PCR) and gold nanoparticles as reporters for the detection of miR-21 and miR-let-7a in human urine. It is the first time that such a biosensor has been applied to the detection of microRNAs in urine. As low as 102-103 copies of miR-21 and 102--104 copies of miR-let-7a added in urine were detectable by the proposed lateral flow assay with great specificity and repeatability (%CVs <4.5%).

Macromolecular crowding agents enhance the sensitivity of lateral flow immunoassays.

Lateral flow immunoassays (LFIA) have a plethora of applications in health, environmental and food sectors for low-cost, simple, and rapid point-of-need testing. Typically, the user only needs to add the sample without any other intervention from sample application to results. A compelling challenge, and a constant pursuit in LFIA is to improve the assay sensitivity without compromising the simplicity and practicality. We report that the addition of water-soluble macromolecular crowding agents leads to an enhancement of the sensitivity, which is attributed to the fact that the exposure of antibodies and micro/nanoparticle conjugates to macromolecularly crowded environment, while migrating through the confining pores of the strip-pads by capillary forces, promotes the interactions that are responsible for analyte recognition and signal generation. The effect was shown by using two of the most widely established LFIA tests worldwide, that is, detection of nucleocapsid protein from SARS-CoV-2 associated with COVID-19 and detection of Strep-A antigen from Streptococcus pyogenes associated with pharyngitis. For immediate demonstration of the sensitivity enhancement, we worked directly on commercially available devices already optimized in terms of reagents and conditions. Of the crowders used, ficoll, Mr 400000, and ficoll, Mr 70000, gave a 5-10-fold improvement of the signal without affecting the background. Because the addition of macromolecular crowding agents is complementary to other strategies of sensitivity enhancement, such as the design of novel labels and the introduction of signal amplification, we anticipate that the proposed modulation will be extended to numerous analytes with a variety of reporters and LFIA configurations.

Mix-and-read method for assessment of milk pasteurization using a smartphone or a common digital camera.

Alkaline phosphatase (ALP) is the most widely used marker of the adequacy of milk pasteurization since it is inactivated at temperatures slightly higher than those required for elimination of pathogens. The cutoff level is 350 mU/L. The approved colorimetric, fluorometric, and chemiluminometric methods require specialized readers with photomultipliers as detectors, and the samples are usually analyzed one-by-one. We developed a low-cost mix-and-read method that exploited a smartphone or a common digital camera as detectors for the chemiluminometric determination of ALP in milk. As samples, we used pasteurized cow and sheep milk spiked with ALP, as well as mixtures of pasteurized and raw (non-pasteurized) milk. Chemiluminescence images acquired by the smartphone or the digital camera were analyzed by the ImageJ software. The limits of detection (LODs), for images captured by the smartphone, were 4.4 mU/L and 11.1 mU/L for cow milk and sheep milk, respectively, while with the digital camera, the respective LODs were 6.2 mU/L and 6.7 mU/L, respectively. The coefficients of variation (CVs) at the cutoff level of 350 mU/L were 8% and 8.5% for the cow and sheep milk, respectively. For images by the digital camera, the CVs were 5.8% and 5% for cow and sheep milk, respectively. The performance of the method is similar to methods that use a microtiter plate and a luminometer for chemiluminescence measurements. Sample pretreatment is not necessary. The microtiter well format combined with detection by a smartphone enables the analysis of multiple samples simultaneously. It is anticipated that the method will prove useful for the rapid assessment of milk pasteurization efficiency in dairy industries, especially in remote areas where expensive instruments are not available. Graphical abstract.

Smartphone-based chemiluminometric hybridization assays and quantitative competitive polymerase chain reaction.

The present report introduces the smartphone as a simple, low-cost detector/imager for chemiluminometric hybridization assays and quantitative competitive polymerase chain reaction (QCPCR). In QCPCR the amplification products from the target and the competitor DNA have identical sizes but differ in a short sequence flanked by the primers. The products are hybridized with their cognate oligonucleotide probes, captured on microtiter wells and detected via an enzyme-catalyzed chemiluminogenic reaction using the smartphone as a detector/imager. We provide, for the first time, data on: (a) the detectability, analytical range and reproducibility of smartphone-based chemiluminometric hybridization assays of double stranded amplification products, (b) the comparison of smartphone-based detection with a conventional digital camera and a luminometer, and (c) the detectability, analytical range and reproducibility of smartphone-based QCPCR in terms of the number of copies of input target sequences in the sample prior to amplification. The limits of detection of the DNA hybridization assay based on the smartphone, digital camera and luminometer were 1.6, 2.4 and 1 pmol L-1. Smartphone-based QCPCR showed an analytical range from 137 to 9 × 105 copies of target DNA.

Paper-based device providing visual genetic signatures for precision medicine: application to breast cancer.

Genome-wide association studies have demonstrated that combinations of single nucleotide polymorphisms (SNPs), rather than individual SNPs, represent genetic signatures that correlate with heterogeneous and complex diseases. In this context, we developed a paper-based device that provides visual detection of a 10-SNP panel as a genetic signature associated with the risk for breast cancer. The method involves multiplex PCR amplification, multiplex extension reaction of allele-specific primers, without prior purification of the amplified sequences, and, finally, capture and visualization of the extension products within minutes on the device. Detection and monitoring are accomplished either by naked eye or by scanning with a common flatbed scanner. The total assay time is ∼ 2 h. The method was evaluated by using 21 clinical samples of known genotypes. The results were fully concordant with the reference method (sequencing). The proposed method is accurate, simple, rapid, and cost-effective. Visual detection does not require specialized instrumentation or highly trained technical personnel. We anticipate that the proposed device will become a useful analytical tool for precision medicine of breast cancer.

Lateral flow test for meat authentication with visual detection.

A new lateral flow assay has been developed for meat authentication. The assay is based on gold nanoparticles and enables visual detection, by naked eye, of meat species-specific DNA sequences. The procedure includes DNA isolation from fresh meat samples, amplification of specific DNA sequences and detection by the lateral flow assay. The proposed assay is rapid and has been applied for the identification of four animal species: horse, pork, beef and sheep. The detection is completed within 25-30 min after amplification. The assay offers high detectability and good selectivity and reproducibility. As low as 0.01% of horse and 0.02% of pork DNA were detectable in binary mixtures by the reported lateral flow device.

Multianalyte quantitative competitive PCR on optically encoded microspheres for an eight-gene panel related to prostate cancer.

Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene. Using PCR as a synthetic tool, a competitor was prepared for each target sequence containing the same primer binding sites as the target but differing in a short segment to enable discrimination by hybridization. The assay involves multiplex amplification of targets and competitors followed by a multiplex hybridization assay for the 16 amplification products. The assay was performed on optically encoded microspheres with oligonucleotide probes attached to their surface. The microspheres were analyzed rapidly (1 min) by flow cytometry. The signal ratio of the target and cognate competitor is a function of the target copy number in the sample prior to amplification. The multiplexing potential of the proposed method is much higher than real-time PCR and other end-point methods since there are 100 sets of commercially available microspheres.

Advances in microRNA analysis.

MicroRNAs (miRNAs) are single-stranded noncoding RNA molecules that act as key regulators of mRNA expression and are emerging biomarkers for disease. Their small size (18-25 nt) presents challenges to molecular recognition, labeling, and signal generation. Recent research activity in this field has aimed at the development of methods for miRNA quantification that combine high detectability, broad dynamic range, practicality, multiplexity, and low cost for prospective applications in diagnostic laboratories. This review article covers the most recent advances in microRNA analysis.

Digital camera and smartphone as detectors in paper-based chemiluminometric genotyping of single nucleotide polymorphisms.

Chemi(bio)luminometric assays have contributed greatly to various areas of nucleic acid analysis due to their simplicity and detectability. In this work, we present the development of chemiluminometric genotyping methods in which (a) detection is performed by using either a conventional digital camera (at ambient temperature) or a smartphone and (b) a lateral flow assay configuration is employed for even higher simplicity and suitability for point of care or field testing. The genotyping of the C677T single nucleotide polymorphism (SNP) of methylenetetrahydropholate reductase (MTHFR) gene is chosen as a model. The interrogated DNA sequence is amplified by polymerase chain reaction (PCR) followed by a primer extension reaction. The reaction products are captured through hybridization on the sensing areas (spots) of the strip. Streptavidin-horseradish peroxidase conjugate is used as a reporter along with a chemiluminogenic substrate. Detection of the emerging chemiluminescence from the sensing areas of the strip is achieved by digital camera or smartphone. For this purpose, we constructed a 3D-printed smartphone attachment that houses inexpensive lenses and converts the smartphone into a portable chemiluminescence imager. The device enables spatial discrimination of the two alleles of a SNP in a single shot by imaging of the strip, thus avoiding the need of dual labeling. The method was applied successfully to genotyping of real clinical samples. Graphical abstract Paper-based genotyping assays using digital camera and smartphone as detectors.

Τwo-panel molecular testing for genetic predisposition for thrombosis using multi-allele visual biosensors.

Thrombosis is considered as the most typical example of multigenic/multifactorial disorder. The three most common genetic risk factors for thrombotic disorders are the G1691A mutation in factor V gene (FV Leiden), the G20210Α mutation in prothrombin gene (FII), and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. An additional panel of biomarkers predisposing for thrombotic events includes the H1299R variant in factor V gene (HR2), A1298C variant in MTHFR gene, the V34L mutation in fibrinogen stabilizing factor XIII (FXIII) gene as well as the 4G/5G polymorphism in plasminogen activator inhibitor type-1 (PAI-1) gene. In this context, we report a novel, rapid and low-cost two-panel diagnostic platform for the simultaneous visual genotyping of the seven mutations (14 alleles). The proposed method comprises the following: (a) a multiplex PCR using genomic DNA isolated from peripheral blood, (b) a multiplex genotyping reaction based on allele-specific primer extension, and (c) visual detection of the genotyping reaction products by means of a multi-allele dipstick-type DNA biosensor, using gold nanoparticles as reporters. The method was applied to 40, previously characterized, and 15 blind clinical samples and the results were 100 % accurate. The proposed assay is simple to perform, requires no specialized and costly equipment, and eliminates multiple pipetting, incubation, and washing steps.

Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene.

Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.

Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis.

BACKGROUND: Screening for "non-deletion" α-chain haemoglobin variants resulting from point mutations or short deletions/insertions has attracted an increased interest during recent years, especially in areas where α-thalassaemia is prevalent. We describe a method utilising high resolution melting analysis for detecting the 13 most common "non-deletion" α-thalassaemia mutations in populations around the Mediterranean and Middle East. METHODS: The method comprises: (1) amplification of a 1087 bp fragment for each of the duplicated α-globin genes (HBA1 and HBA2) flanking all 13 mutations using a common forward primer and different reverse primers specific for HBA1 and HBA2, respectively; (2) nested amplification of three fragments in HBA2 flanking 10 mutations and two fragments in HBA1 flanking 5 mutations; (3) High resolution melting analysis of the amplicons using a LightScanner Instrument and LC Green. RESULTS: All 13 "non-deletion" α-chain haemoglobin variants were successfully detected by high resolution melting analysis. All heterozygote samples and eight out of 10 available homozygotes were clearly differentiated from each other and from wild type in the same amplicon. Although not all homozygote samples were distinguishable from wild type samples, this should not present a problem in a clinical setting since all DNA results should be evaluated alongside the haematological and (if relevant) clinical findings in each case. CONCLUSIONS: The 13 "non-deletion" α-chain haemoglobin variants were successfully genotyped by high resolution melting analysis using LightScanner instrument and LCGreen Plus saturating dye. High resolution melting analysis is an accurate mutation scanning tool, advantageous as a closed-tube method, involving no post-PCR manipulations and requiring only around 5 min post-PCR analysis.

Multi-allele DNA biosensor for the rapid genotyping of 'nondeletion' alpha thalassaemia mutations in HBA1 and HBA2 genes by means of multiplex primer extension reaction.

BACKGROUND: Alpha-thalassaemia is an autosomal recessive disorder characterized by defective production of the alpha chain of haemoglobin. It is caused mainly by deletions of one or both of the duplicated alpha-globin genes on chromosome 16, and/or by nucleotide variations, known as "nondeletion" mutations. Definition of the alpha globin genotype in carriers supports genetic counselling, and in patients with Hb H disease is useful to predict prognosis and management options. Here, we report a method that facilitates direct detection by naked eye of the 13 most common "nondeletion" alpha-globin gene mutations in populations around the Mediterranean and Middle East. METHODS AND RESULTS: The method comprises (i) PCR amplification of a single 1087 bp fragment for each HBA1 and HBA2 gene (separately); (ii) multiplex primer extension reaction of just 10 cycles, using unpurified amplification product as a template, to incorporate biotin into those allele-specific primers that extend and, finally, (iii) visual detection of the reaction products within minutes by the dipstick biosensor. The method was evaluated by analysing 105 samples of known genotypes and the results were found fully concordant with those obtained by the reference methods. CONCLUSIONS: The proposed assay is particularly suited for small molecular-diagnostic laboratories with a limited budget and a low-to-medium sample volume. In addition this platform represents a very simple and useful genotyping tool to support gene scanning methods whenever nucleotide variations have to be specified.

Olive oil DNA fingerprinting by multiplex SNP genotyping on fluorescent microspheres.

Olive oil cultivar verification is of primary importance for the competitiveness of the product and the protection of consumers and producers from fraudulence. Single-nucleotide polymorphisms (SNPs) have emerged as excellent DNA markers for authenticity testing. This paper reports the first multiplex SNP genotyping assay for olive oil cultivar identification that is performed on a suspension of fluorescence-encoded microspheres. Up to 100 sets of microspheres, with unique "fluorescence signatures", are available. Allele discrimination was accomplished by primer extension reaction. The reaction products were captured via hybridization on the microspheres and analyzed, within seconds, by a flow cytometer. The "fluorescence signature" of each microsphere is assigned to a specific allele, whereas the signal from a reporter fluorophore denotes the presence of the allele. As a model, a panel of three SNPs was chosen that enabled identification of five common Greek olive cultivars (Adramytini, Chondrolia Chalkidikis, Kalamon, Koroneiki, and Valanolia).

Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters.

There is a growing interest in the development of biosensors in the form of simple lateral flow devices that enable visual detection of nucleic acid sequences while eliminating several steps required for pipetting, incubation and washing out the excess of reactants. In this work, we present the first dipstick-type nucleic acid biosensors based on quantum dots (QDs) as reporters. The biosensors enable sequence confirmation of the target DNA by hybridization and simple visual detection of the emitted fluorescence under a UV lamp. The 'diagnostic' membrane of the biosensor contains a test zone (TZ) and a control zone (CZ). The CZ always fluoresces in order to confirm the proper function of the biosensor. Fluorescence is emitted from the TZ, only when the specific nucleic acid sequence is present. We have developed two general types of QD-based nucleic acid biosensors, namely, Type I and Type II, in which the TZ consists of either immobilized streptavidin (Type I) or immobilized oligodeoxynucleotides (Type II). The control zone consists of immobilized biotinylated albumin. No purification steps are required prior to the application of the DNA sample on the strip. The QD-based nucleic acid biosensors performed accurately and reproducibly when applied to (a) the visual detection of PCR amplification products and (b) visual genotyping of single nucleotide polymorphisms (SNPs) in human genomic DNA from clinical samples. As low as 1.5 fmol of double-stranded DNA were clearly detected by naked eye and the dynamic range extended to 200 fmol. The %CV were estimated to be 4.3-8.2.