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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.

A High Fundamental Frequency QCM Immunosensor for Tuberculosis Detection

AWSensors biosensor for tuberculosis detection

 

Authors: Ángel Montoya, Carmen March, Yeison J. Montagut, Maria J. Moreno, Juan J. Manclus, Antonio Arnau, Yolanda Jiménez, Marisol Jaramillo, Paula A. Marin, Robinson A. Torres

Journal:  Current topics in Medicinal Chemistry (2017)

Background: Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization.

Method & Material: Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen.

Results & Discussion: For the development of the HFF piezoelectric immunosensor, 100 MHz AWSensors quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum.

You may review the full paper here

Bio-Logic AWS-A20 at Biosensing Conference

AWSensors at the 5th International Conference on Biosensing technology


AWSensors technology will be at the  5th International Conference on Bio-sensing technology to be held at Riva del Garda, Italy, from May 7th to 10th. We are pleased to invite you to see our biosensing platforms at the booth of Bio-Logic, our OEM distributor.

AWS A20-F20 platform is the most flexible and sensitive QCM-D system in the market. It is able to work with low and high frequency QCM sensors and SAW sensors. It also ensures high sensitivity because of a novel method of characterization that keeps noise at very low level in spite of working at high frequencies by using AWS-HFF sensors (100-150 MHz). AWSensors also provides QCM and SAW sensors and a wide range of cells and accessories to customize experiments and tests according to specific conditions required by scientists.

Therefore, AWS A20 system provides you with big competitive advantages:

–       High flexibility, capable to work with any acoustic wave sensor; modular system capable to be upgraded with more channels after purchase (up to 4)

–       High sensitivity

–       Customization, with accessories to make measurements in specific conditions

–       Price convenience

Do not miss the opportunity to check the advantages of our technology at the booth of Bio-Logic and meet our Application Scientist, Mrs. María García.

Dynamic Resolution of Ion Transfer in Electrochemically Reduced Graphene Oxides Revealed by Electrogravimetric Impedance

Authors: Hamza Goubaa, Freddy Escobar-Teran, Ibtissam Ressam, Wanli Gao, Abdelkrim el Kadib, Ivan T. Lucas, Mustapha Raihane, Mohammed Lahcini, Hubert Perrot, Ozlem Sel. Sorbonne Université, UPMC, Univ Paris 06, CNRS, France. Laboratoire Interfaces et Systèmes Électroquimiques; Université Cadi Ayyad, Faculté des Sciences et Techniques, Laboratoire de Chimie Organométallique et Macromoléculaire Matériaux composites, Marrakech, Morocco.

Journal:  The Journal of Physical Chemistry (2017)

To accompany the search for optimal materials in electrochemical supercapacitors, appropriate characterization tools to assess key parameters of newly developed electrodes are required. Here we demonstrate the capabilities of ac-mode electrogravimetry to study in details the capacitive charge storage mechanisms in electrochemically reduced graphene oxide (ERGO) thin films electrodes. The coupling of electrochemical impedance spectroscopy (EIS) with fast quartz crystal microbalance (QCM) complements classical electrochemical quartz crystal microbalance (EQCM) by capturing here the dynamics of the electroadsorption process, identifying charged moieties and detecting solvation effects. We evidenced the co-electroadsorption of two types of cationic species (fully and partially hydrated cations) in the potential range studied and the indirect intervention of free solvent molecules. Further kinetic information on electroadsorption is also obtained which leads to a full deconvolution of the global EQCM response at both gravimetric and temporal level. Through a fine analysis of the interactions of different cations of period one elements, Li+, Na+ and Kwith ERGO, we evidenced the critical role of solvation processes on the kinetics of electroadsorption, and provided an experimental proof to the phenomena that smaller ions are more tightly bound to their water molecules such that an inverse relationship exists between the dehydration energy and the ion size. Such gravimetric and dynamic subleties are unreachable with classical tools and with EQCM method alone which permits us to suggest the ac-mode electrogravimetry as a baseline diagnostic tool to explore charge transfer mechanisms at the nanoscale.

Further sensitivity enhancement of HFF-QCM immunosensors for pesticides

Authors: C. March, J.V. García, R. Fernández, Y.Jiménez, A. Arnau. A.Montoya

Event: 4th International Conference on Biosensing Technology, Lisbon (2015)

Recently, High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) immunosensors have succesfully been developed. Therefore, sensitivity of QCM biosensors is no longer a drawback. Taking advantage of this previous work, we have developed a renewed highly sensitive HFF piezoelectric immunosensor using carbaryl insecticide as a model analyte for pesticide detection. To this purpose, 100 MHz quartz crystal sensors were used as the transducer elements of the biosensor and a monoclonal antibody-based competitive immunoassay was integrated as the sensing specific bio-recognition event. The biosensing interface was improved by employing mixed self-assembled monolayers (mSAMs) of alkane thiols as intermediate layers for surface functionalization. This approach allowed the covalent attachment of the assay conjugate (20.0 µg mL-¹ of BSA-CNH conjugate) onto the gold electrode surface in a more orderly and stable way than with simple SAMs. A very low concentration (1.0 µg mL-¹) of LIB-CNH45 monoclonal antibody was used for the competitive immunoassays. All immunosensor assays were performed in the AWS-A10 test platform from AWSensors. In terms of analytical performance, the new carbaryl HFF-QCM immunosensor showed higher sensitivity than the previously developed one, with analytical parameters very close to those of the most sensitive reported ELISA for carbaryl.

CLICK HERE TO DOWNLOAD THE POSTER about this work

Comparison between High Fundamental Frequency Quartz Crystal Microbalance and Love Mode Surface Acoustic Wave devices in the detection of Carbaryl pesticide

Authors: J.V. García, M.I. Rocha, C. March, P. García, L.A. Francis, A. Montoya, A. Arnau, Y. Jiménez

Event: Acoustic Sensors in analytical and Biophysical Studies, 29-30 August 2013, IMBB-FORTH, Heraklion, Crete.

 

In this work two acoustic technologies: High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) and Love Mode Surface Acoustic Wave (LM-SAW) have been compared for the detection of Low Molecular Weight (LMW) compounds in terms of Sensitivity and LOD. The results have also been compared with those obtained with other techniques: Traditional QCM, Surface Plasmon Resonance (SPR) and Enzyme-Linked ImmunoSorbent Assay (ELISA). Carbaryl pesticide was chosen as model analyte because it had been used as a reference LMW compound by those mentioned techniques.

AWS-A10 research platform (AWSensors, Spain) was used to perform the experiments. This platform allowed for a comparison of both devices measured by the same characterization system under similar experimental conditions.

The results achieved with LM-SAW and 100Mhz HFF-QCM were in the same order of magnitude. The achieved value sensitivity (I50 value) and LOD (I90 ) were around 0.31 µ/L and 0.09 µ/L, respectively, for LM-SAW device, and around 0’66 µ/L and 0’14 µ/L, respectively, for HFF-QCM device. Sensitivities and LODs with these novel developments on acoustic technologies improve SPR and traditional QCM technologies, and approach ELISA’s ones.

Review the poster about this experiment by clicking here:

Póster about AWS-HFF sensors for detection of Carbaryl pesticide showed at Crete 2013