Tag Archive for: DNA

Publication on AWSensors technology

Portable Surface Acoustic Wave device platform coupled with a paper-based capillary fluidics for real-time biosensing applications

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Authors: Angelos Ntimtsas and Electra Gizeli

Journal: Chemical Society Sensors and Actuators A: Physical

Abstract: Surface acoustic wave (SAW) sensors have emerged as prominent real-time, label-free biosensors with diverse applications in immunoassays and nucleic acid detection. However, widespread adoption in diagnostics has been impeded by challenges such as miniaturization of instrumentation, microfluidics fabrication and high attenuation in liquid environments. In this study we address these limitations by presenting a portable platform capable of real-time monitoring of a SAW microarray chip comprising up to four sensing channels, combined with a novel paper-based capillary flow channel. For the fabrication of a portable, automated and versatile sensor platform, we integrated a microcontroller, RF components, and a micropump for flow control. Calibration procedures ensured accurate measurements for both amplitude and phase, with a low drift rate (0.72 mdB/h and 1.00 mdeg./h, respectively) and high resolution (2.10 mdB and 6.10 mdeg.), indicating stable operation with minimal interface electronics. The capillary flow channel, implemented by confining the fluid on the sensing surface using a nitrocellulose strip, facilitated laminar and continuous sample flow with minimum damping of the acoustic signal. The sensor system was evaluated with two SAW devices at 100 and 200 MHz using glycerol dilutions within the range of 1% and 70%, demonstrating real-time monitoring capabilities and linear correlations between amplitude and phase changes. To prove the biosensing and clinical validity of the SAW newly developed system, DNA adsorption on a poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) functionalized SAW-surface was demonstrated with a detection limit of 0.001 μg/mL. Our study demonstrates the feasibility of a portable SAW sensor system coupled with a low-cost and replaceable capillary flow channel for real-time monitoring and biomolecular detection.

The full article can be accessed here.

Scientific publication

High-Frequency Quartz Crystal Microbalance and Dual-Signaling Electrochemical Ratiometric Assays of PTP1B Activity Based on COF@Au@Fc Hybrids

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Authors: Shuping Liu, Qingqing Zhang, Xiaohua Zhang, Cuicui Du, Shihui Si, and Jinhua Chen

Journal: Analytical Chemistry

Abstract

The abnormal expression of protein tyrosine phosphatase 1B (PTP1B) is highly related to several serious human diseases. Therefore, an accurate PTP1B activity assay is beneficial to the diagnosis and treatment of these diseases. In this study, a dual-mode biosensing platform that enabled the sensitive and accurate assay of PTP1B activity was constructed based on the high-frequency (100 MHz) quartz crystal microbalance (QCM) and dual-signaling electrochemical (EC) ratiometric strategy. Covalent–organic framework@gold nanoparticles@ferrocene@single-strand DNA (COF@Au@Fc-S0) was introduced onto the QCM Au chip via the chelation between Zr4+ and phosphate groups (phosphate group of the phosphopeptide (P-peptide) on the QCM Au chip and the phosphate group of thiol-labeled single-stranded DNA (S0) on COF@Au@Fc-S0) and used as a signal reporter. When PTP1B was present, the dephosphorylation of the P-peptide led to the release of COF@Au@Fc-S0 from the QCM Au chip, resulting in an increase in the frequency of the QCM. Meanwhile, the released COF@Au@Fc-S0 hybridized with thiol/methylene blue (MB)-labeled hairpin DNA (S1-MB) on the Au NPs-modified indium–tin oxide (ITO) electrode. This caused MB to be far away from the electrode surface and Fc to be close to the electrode, leading to a decrease in the oxidation peak current of MB and an increase in the oxidation peak current of Fc. Thus, PTP1B-induced dephosphorylation of the P-peptide was monitored in real time by QCM, and PTP1B activity was detected sensitively and reliably using this innovative QCM-EC dual-mode sensing platform with an ultralow detection limit. This platform is anticipated to serve as a robust tool for the analysis of protein phosphatase activity and the discovery of drugs targeting protein phosphatase.

You may read the full paper here.

Scientific publication

Real-time monitoring of dephosphorylation process of phosphopeptide and rapid assay of PTP1B activity based on a 100 MHz QCM biosensing platform

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Authors: Shuping Liu, Qingqing Zhang, Xiaohua Zhang, Cuicui Du, Jinhua Chen, Shihui Si

Journal: Talanta

Abstract

The misregulation of protein phosphatases is a key factor in the development of many human diseases, notably cancers. Here, based on a 100 MHz quartz crystal microbalance (QCM) biosensing platform, the dephosphorylation process of phosphopeptide (P-peptide) caused by protein tyrosine phosphatase 1B (PTP1B) was monitored in real time for the first time and PTP1B activity was assayed rapidly and sensitively. The QCM chip, coated with a gold (Au) film, was used to immobilized thiol-labeled single-stranded 5′-phosphate-DNAs (P-DNA) through Au–S bond. The P-peptide, specific to PTP1B, was then connected to the P-DNA via chelation between Zr4+ and phosphate groups. When PTP1B was injected into the QCM flow cell where the P-peptide/Zr4+/MCH/P-DNA/Au chip was placed, the P-peptide was dephosphorylated and released from the Au chip surface, resulting in an increase in the frequency of the QCM Au chip. This allowed the real-time monitoring of the P-peptide dephosphorylation process and sensitive detection of PTP1B activity within 6 min with a linear detection range of 0.01–100 pM and a detection limit of 0.008 pM. In addition, the maximum inhibitory ratios of inhibitors were evaluated using this proposed 100 MHz QCM biosensor. The developed 100 MHz QCM biosensing platform shows immense potential for early diagnosis of diseases related to protein phosphatases and the development of drugs targeting protein phosphatases.

You may read the full paper here.

Publication on AWSensors technology

Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification

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Authors: Nikoletta Naoumi, Monica Araya-Farias, Maria Megariti, Lucile Alexandre, George Papadakis, Stephanie Descroix, and Electra Gizeli

Journal: Analyst

Abstract

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients’ crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

You may read the full paper here.

Publication on AWSensors technology

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy

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Authors: Nikoletta Naoumi, Kleita Michaelidou, George Papadakis, Agapi E. Simaiaki, Román Fernández, Maria Calero, Antonio Arnau, Achilleas Tsortos, Sofia Agelaki, and Electra Gizeli

Journal: ACS Sens. (2022)

Abstract

Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically “lossy” liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.

 

You may read the full paper here.

Liqbiopsens project

AWSENSORS HOSTS THE 24M MEETING OF LIQBIOPSENS EUROPEAN PROJECT

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February 1st 2018

AWSensors host the 24-Month Meeting of LIQBIOPSENS EU project

The meeting of all the project partners was held in Valencia, Spain, at the headquarters of AWSensors and Sistemas Genómicos, where significant advances were presented between the project partners and important decisions were taken to successfully continue pursuing the project goals. Another whole year is still required to complete the project.

The consortium for the development of this project is composed of the following institutions: Servicio Andaluz de Salud (Spain), Université Catholique de Louvin (Belgium),  Foundation for Research and Technology Hellas (Greec), DestiNA Genomics (UK, Spain), Sistemas Genómicos (Spain) and AWSensors (Spain). AWSesensor.

If you wish to know more about the LIQBIOPSENS project, visit the project website at http://liqbiopsens.com.

 

Biotechnology business

AWSENSORS RAISED 1M€ INVESTMENT

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July 27th 2017

Swedish and Spanish companies invested in AWSensors seeing a growth opportunity.

AWSensors raised 1M€ investment round to strengthen its position in scientific market and enter the healthcare tech market, one of the most competitive markets. The company though has convinced Swedish and Spanish investors that the potential payoff is worth the risk.

Firms entering into the investment round were BAble Capital, a venture capital firm that aims to invest in technology-based companies from Spanish universities and research centers, and Tech Transfer UPV, a venture capital firm created specifically to transfer technology developed at the Spanish university, Polytechnic University of Valencia. But also a consortium of Swedish companies invested in AWSensors seeing a growth opportunity. Other Spanish companies like PolymerChar, Keodes or Citrosol are also among the shareholders.

Prof. Arnau, founder of AWSensors

AWSensors, led by Prof. Antonio Arnau, expects to speed up innovation processes to launch new scientific equipments to meet demand from scientists and industry R&D but also to develop cutting-edge healthcare technology to be applied in personalized medicine.

The aim of these new developments is to get a blood test meant to catch cancer when it’s most treatable, before patients show symptoms, by detecting fragments of DNA shed by tumors.  A new liquid biopsy system will allow for detection of this circulating DNA and its mutations and will be an essential equipment to help doctors diagnose, monitor and treat cancer patients in a personalized way.

To hit that goal, AWSensors got another 1M€ from European Comission through two H2020 European projects:

  • LiqBiopSens project, that is coordinated by AWSensors and developed together with companies and institutions from Spain, Belgium, UK and Greece.
  • Catch-u-DNA project, which is carried out with partners from Germany, France, Israel, Greece and Spain.

These projects as a whole were funded with 5.7M€ by European Comission.

AWSENSORS CONSIGUE UNA INVERSIÓN DE 1 MILLÓN DE EUROS

Nuevos inversores de Suecia y España entran en el accionariado de la compañía

AWSensors ha conseguido despertar el interés de inversores nacionales e internacionales y realizar una ronda de 1 millón de euros. En la operación de inversión han participado BeAble Capital, sociedad gestora que tiene como objetivo invertir en empresas de base tecnológica con origen en universidades y centros de investigación españoles, y Tech Transfer UPV, fondo creado específicamente para trasladar al mercado tecnología desarrollada en la Universidad Politécnica de Valencia. Pero también un consorcio de empresas tecnológicas suecas ha apostado fuerte por entrar como inversor en AWSensors. Además, empresas valencianas como PolymerChar, Keodes o Citrosol están también entre los accionistas.

Esta inversión permitirá a AWSensors consolidarse en el mercado científico internacional donde comercializa una nueva tecnología patentada de sensores para análisis de interacciones moleculares en tiempo real que utilizan investigadores de Europa, Estados Unidos y Asia en los campos de ciencias de la vida y nuevos materiales.

La compañía, liderada por el Prof. Antonio Arnau, espera invertir en nueva infraestructura de fabricación y captar nuevo talento para su plantilla. El objetivo es acelerar el proceso de innovación para sacar al mercado nuevos equipos con mayor productividad y automatización e iniciar nuevos desarrollos para el mercado de salud, ya que la tecnología de AWSensors podrá ser aplicada en medicina personalizada.

Uno de los desarrollos previstos para el mercado sanitario es un equipo para la detección precoz de cáncer colorrectal y su monitorización sin necesidad de entrar al quirófano para realizar una biopsia del tumor. Servirá con un análisis de sangre cuyos resultados se podrían obtener en una hora. La detección en este caso se basa en el ADN que libera el tumor en el organismo. El nuevo sistema permitirá detectar ese ADN circulante y las mutaciones que sufre asociadas al cáncer. Este análisis, denominado biopsia líquida, permitirá un control sencillo del paciente y facilitará por tanto la adaptación del tratamiento de forma personalizada.

Liqbiopsens project

Este nuevo equipo se está desarrollando junto a otras empresas e instituciones de España, Bélgica, Reino Unido y Grecia dentro del proyecto LiqBiopSens financiado por la Comisión Europea dentro del programa H2020. Un proyecto que ha inyectado medio millón de euros a AWSensors.

Paralelamente a este desarrollo, AWSensors participa en otro proyecto europeo, Catch-u-DNA que investiga una nueva técnica para mejorar la sensibilidad de la tecnología en la detección del ADN circulante. Este proyecto, que se realiza con socios de Alemania, Francia, Israel, Grecia y España, ha supuesto el ingreso de medio millón de euros más en la compañía.

En total, la Comisión Europea ha dotado con 5’7 millones de euros a estos dos proyectos de investigación cuyos resultados impulsarán el crecimiento de AWSensor

Catch-u-DNA project towards a new technology for molecular diagnosis

New project to validate an easier and faster technique of molecular diagnosis applied to cancer by using quartz sensors

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July 7th 2017

In 2015 AWSensors already got the European project “LiqBiopSens” to develop a liquid biopsy platform for early detection and monitoring of colorectal cancer. Now, the company will work in the European  project “CATCH-U-DNA”, worth 3.4 million euros. CATCH-U-DNA poses a new concept in biophysics and molecular diagnosis based on acoustic sensing of DNA in serum. It aims to provide a novel technology for the ultrasensitive detection of circulating-tumor DNA in serum without PCR amplification.

“CATCH-U-DNA” project was granted under the Horizon 2020 FET-OPEN call, aimed at financing radically new initiatives that can have a long-term economic and social impact. Only the most cutting-edge technology projects are chosen. It is one of the most competitive European programs, where less than 4 per cent of the ideas presented are funded. The project is coordinated by Prof. Electra Gizeli, leader of the Biosensors group in the Institute of Molecular Biology and Biotechnology of the Greek Foundation for Research and Technology-Hellas (FORTH).

Detection of lung and colorectal cancer

The ultimate goal of “CATCH-U-DNA” is to validate a new, simpler and cheaper technology for the detection of genetic markers so that personalized medical diagnosis can be performed in a more precise, easy and affordable way than current technologies.

The project faces the ambitious challenge of testing that radically new technology in the early diagnosis of cancer by detecting the most common genetic mutations that cause colorectal cancer and lung cancer. In fact, it includes not only experimental research but also clinical trials.

In the future, it will be possible to incorporate this new technique into portable equipment and will therefore allow on-site analysis applied to personalized medicine in developed countries or in areas without laboratory infrastructure such as underdeveloped countries.

New technique easier, more affordable and more precise

The amount of mutated DNA in a sample is generally very low, which greatly hampers its detection and characterization. Most of the current methods of detection are based on the technique known as PCR (polymerase chain reaction), which allows amplifying a fragment of DNA by obtaining millions of copies, thus, its detection in the lab is feasible. However, this technique involves a complex and expensive procedure prior to detection. In addition, it may cause a deviation if the amplification is not developed properly.

The “CATCH-U-DNA” technology promises to overcome these limitations and allow for the genetic analysis of human samples in an easier, more affordable and more precise way. The aim is to manufacture an ultra-sensitive device capable of detecting DNA in human samples without any previous amplification procedure even though the amount of that DNA is minimal. Specifically, the goal is the detection of circulating DNA, fragments from the tumor cells found in the blood.

The new technique will use high frequency quartz crystal sensor arrays that allow for a real-time, label-free monitoring, in combination with a revolutionary DNA identification system based on the hydrodynamic molecular properties. This approach will allow to push the detection limit down obviating the amplification.

7 companies and organizations from 5 different countries

The “CATCH-U-DNA” project will be developed over 3 years. Experts in molecular biology, physics, chemistry, nanomaterials, biosensors and microfluidics from Spain, Germany, France, Greece and Israel are involved. They are from the Greek Foundation for Research and Technology Hellas (FORTH), which is the project coordinator, and from AWSensors; the Autonomous University of Madrid, in Spain; the University of Crete in Greece; the Curie Institute in France; the Ben Gurion University of Negev in Israel and the German company Jobst Technologies.

 

honey

AWSensors technology to develop a DNA biosensor for detection of honey adulteration

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May 15th 2017

AWSensors technology will be used in a new research project funded by Spanish Government (Retos Investigación 2017-2019) to develop a DNA biosensor for detection of honey adulteration. Honey companies are interested in this biosensor because new and cheaper analytical methods are required to meet the quality controls set by European Comission.

One of the priority challenges of the European Union (EU) is “Quality and Security in Food”. Food adulteration is a topic of interest in several fields: health care, legal (since it is a fraud) and economic (since it generates unfair competition). In this context, honey is one of the most commonly adulterated food, which generates a great deal of economical problems in apiarian production and comercialization sector. This situation affects directly to Spain, since is the most important EU country in honey production and comercialization. Nowadays, honey adulteration is made, mainly, by using vegetal siropes, resulting in an adulterated product similar in taste to natural honey, but fraudulent, and including substances that consumer unknowingly ingests. Therefore, the European Comission is promoting the development of new analytical methods which complement or replace the already existing ones.

 

The main drawbacks of the already existing techniques are:

  1. There is no a unique technique which allows to identify, in a reliable way, an adulteration; therefore, in order to be conclusive, several analytical determinations are needed;
  2. They are only available in central laboratories, which directly affects to the companies quality control process, slowing it down and rising its costs;
  3. They requiere high qualified staff;
  4. They requiere long time analysis periods (hours)
  5. They have not enough resolution to detect the target substances sometimes.

 

 

DNA biosensors are becoming very promising in the field of security and quality food control, since they are easy handling, reliable, fast and low cost. The proposed technology is based on the use of acoustic sensors coated with functionalized nanostructures which allow to greatly increase the Limit of Detection (LOD) of the DNA of the substances used in honey adulteration.

 

In this scenario, techniques based on DNA biosensors are becoming very promising in the field of security and quality food control, since they are easy handling, reliable, fast (analysis periods: minutes) and low cost. In this research project, the use of a novel technology in the field of food control adulteration is proposed. This technology is based on the use of acoustic sensors coated with functionalized nanostructures which allow to greatly increase the Limit of Detection (LOD) of the DNA of the plant substances used in honey adulteration. The use of those mentioned nanostructures generates a mechanic-acoustic amplification effect and, moreover, allow to separate the sensor transduction mechanism from the biochemical recognition process (DNA hybridization). The expected result is an increase of more than one order of magnitude in the sensor response when comparing it with the response of a sensor without the nanostructure coating.

The research proposed in this project deals with new challenges:

  1. The use of a new recognition method based on DNA detection;
  2. The use of nanostructures which provide a mechanic-acoustic amplification and a separation of the transduction mechanism from the biochemical recognition process;
  3. The use of a new technique for sample dispensing based on an in-batch method.

 

To deal with these challenges, a multidisciplinary research team of experts in micro and nano electronics, advanced materials and biotechonology is required to guarantee the succes of the project. AWSensors will collaborate with this team of scientists from the Polytechnic University of Valencia, in Spain, (Bioengineering Research and Innovation Center and University Institute of Food Engineering for Development) and University Pierre et Marie Curie, in France (Laboratoire interfaces et systémes electroquimiques). Other honey companies such as Apisol, Honeygreen, Cooperativa Apícola de España, Granalbe and Primo Mendoza are interested in the results of the project.

A liquid biopsy platform combining a high fundamental frequency QCM device with dynamic chemistry for detecting mutations in circulating DNA

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Authors: A. Grammoustianou, G. Papadakis, M. Tabraue, J.J. Díaz-Mochon, R. Fernández, J.V. García, A. Arnau, E. Gizeli. AWSensors S.L., Institute of Molecular Biology and Biotechnology- FORTH, University of Crete, Destina Genomics S.L., Centro de Investigación e Innovación en Bioingeniería – Universidad Politécnica de Valencia

Event:  5th International Conference on Bio-sensing Technology, Riva del Garda, Italy (2017)

In the past decade, the analysis of circulating tumour DNA (ctDNA) in blood has been a major breakthrough; ctDNA has been proposed as a priceless source for cancer diagnostic, prognostic and treatment monitoring through a new methodology known as “Liquid Biopsy”. This study presents a novel diagnostic method for the acoustic detection of KRAS mutations in ctDNAs based on: (1) DNA analysis by “dynamic chemistry” that utilizes aldehyde modified nucleobases (SMART) and abasic peptide nucleic acids (DGL probes) capable for the errorfree detection of nucleic acids and their mutations; and, (2) a high fundamental frequency (100 MHz) acoustic wave microsensor (AWS HFF-QCM) that allows the accurate, inexpensive, label-free and real time monitoring of the “dynamic chemistry”. Surface-immobilized DGL probes on the AWS HFF-QCM device are used to detect ctDNAs of wild type and mutated KRAS variants. Upon hybridization of the DGL probe with its target ssDNA, a duplex is formed where biotin- tagged SMART bases can lock in front of the position under interrogation; streptavidin binding detected in a follow-up step confirms the presence of the SMART bases. The use of DGL probes in combination with an isothermal DNA amplification step RPA) have allowed the sensitive and specific recognition of single mismatches in KRAS genes in less than 1 hour. This work presents a unique and novel technology that can emerge as a promising tool in the field of cancer diagnostics.

Liquid Biopsy detection protocol

Schema for complete detection protocol:

(A) Extracted DNA containing mutant (red) and wild type DNA fragments (black) are enzymatically amplified.

(B) Denatured amplicons are hybridized on surface immobilized DGL probes. Chemical locking of a specific tagged SMART base takes place only in the appropriate position.

(C) Incorporated SMART bases are recognized by streptavidin and monitored in real-time during an acoustic measurement.