Tag Archive for: DNA

Publication on AWSensors technology

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

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

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

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

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

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

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.

DNA

H2020 Project LiqBiopSens: A new liquid biopsy platform for early detection of colorrectal cancer

December 10th 2015

We are proud to announce that the European Comission awarded a grant of a total value of 2’7M euros to our project LIQBIOPSENS for early detection of colorectal cancer by carrying out liquid biopsy. The grant was awarded within Horizon2020 and specifically in the call entitled “ICT-28-2015: Cross cutting ICT Key Enabling Technologies”.

 

AWSensors will coordinate this H2020 project during the next 3 years (2016-2018). The overall aim of this project is the further development and validation in real settings of a novel diagnostic platform for the early and fast detection of circulating tumor DNA (ctDNA) and their KRAS and BRAF mutations associated to colorectal cancer through blood samples.

Liquid Biopsy platform LiqBiopSens features

The main features of LiqBiopSens are:

  1. Reliability (detection rates vary from 95-100 %)
  2. Low-Cost
  3. Sensitivity (in the zM range)
  4. Multiplexing capabilities (analysis of 27 KRAS and BRAF mutations simultaneously)
  5. Short analysis time (30-60 min.)
  6. User-friendly interface
  7. Flexibility

LIQBIOPSENS platform is based on the integration of two novel complementary technologies:

  • DGL© technology property of DestiNA Genomics Ltd, capable of delivering faster, more error-free detection of DNA and their mutations than current enzyme-based detection systems, making ‘false positive’ results a thing of the past.
  • A novel high resolution acoustic wave microsensor technology property of AWSensors, that allows an accurate, inexpensive, label-free, direct and real-time transduction method to quantitatively evaluate the results of the application of the mentioned DGL© technique.

However, solution proposed by the LIQBIOPSENS project relies on the multidisciplinary integration of different key enabled technologies. Accordingly, LiqBiopSens will be accomplished by AWSensors and Destina Genomics in collaboration with the following partners:

Liquid Biopsy platform LiqBiopSens concept

The operating concept of our project is described in the following representative scheme:

Early detection of colorrectal cancer by liquid biopsy

ctDNA amplicons produced in chamber (1) are denatured and transferred to the sensors surface via   microfluidics (2) where hybridization with DGL probes takes place; mutation‐discrimination is carried out during the injection (3) of novel probes which click selectively on the target DNA (4) while acoustic detection exploits functionalized nanoparticles (5).

Colorectal cancer, the second most common cause of cancer death

The most frequently occurring forms of cancer in the EU are colorectal, breast, prostate and lung cancers. In men, lung cancer is the most frequent cause of cancer death, while in women, it is breast cancer. In both men and women, colorectal cancer is the second most common cause of cancer death.

The “gold standard” for identifying the type and extent of a cancer is a tissue biopsy, in which a small sample of tissue is taken from the suspected tumour and histologically examined. While this procedure can provide important information about the patient disease, tissue biopsy can be painful, represents a single snap-shot in time, is subjected to significant selection bias and if the tumour tested was first detected by palpation or imaging, it may already be so large that the disease is well advanced. Furthermore, when the tumour tissue is removed or it is inaccessible, these genotyping techniques are impossible. Therefore, cancer remains a ghost disease when primary tumours are removed through surgery, meaning that there are not tools to assess the efficiency of treatments or prevent metastasis.

Liquid Biopsy, a revolution in the fight against cancer

It has long been known that solid tumors release DNA in the blood and the load of circulating tumor DNA (ctDNA) has been correlated to staging and prognosis. However, only recent advances in the sensitivity and accuracy of DNA analysis have allowed for genotyping of somatic changes found in tumors by interrogating ctDNA. This technique is known as ‘Liquid Biopsy’ and it is based on the analysis of a biomarker that can be easily isolated from many body fluids (blood, saliva, urine, ascites, pleural effusion, etc.).

 

Successful implementations of liquid biopsy techniques to track tumor dynamics in real time and for the early detection, such as LIQBIOPSENS, are going to revolutionise how clinicians fight against cancer.