Highly efficient biofunctionalized nanofluidic system for real-time biomarker sensing with molecular enrichment and spatiotemporally resolved kinetics – DEPSLIT

To reach our objectives, the workload is divided into 4 work packages.
WP 1: The first objective of the project is the fabrication of biofunctionalized nanoslits. The sensor will consist of probe molecules patterned within the nanoslit and target detection is then to be carried out by fluorescence measurement of bound fluorescent targets. We will specially address two general problems encountered in microfluidic-based biosensing in the specific case of our nanofluidic approach. How do we properly block the non-reactive surfaces of the nanochannels to avoid non-specific adsorption and target depletion? How do we immobilize different probes on the same chip for multiplexed detection?
WP 2: This work package targets the affinity-based biosensing application where modeling tools are required to extract binding constants from experimental sigmoids. This task will be addressed by modeling the nanoslit with FEM, and then by developing a simplified simulation tool, taking into account our specific non-diffusion limited regime, in order to efficiently carry out parametric fitting of the experimental data and extraction of kinetic constants.
WP 3: We will study the efficiency of electrokinetically driven flow and the effect of the molecular enrichment scheme by molecular damming effect of eDEP with nanoconstrictions on the enhancement of mass transport.
WP 4: The last work package intends to demonstrate the interest of nanoslit biosensors with implemented target enrichment scheme for (1) kinetic studies and (2) ultra-high sensitivity detection of relevant biomarkers. For this purpose, we will benchmark our biosensor by directly comparing it to other affinity-based biosensors (i.e. SPR and QCM) and indirectly to state-of-the-art ultra-high sensitive biosensors.

Results obtained within each WP:
WP 1:
- The consortium has successfully implemented the fabrication process of the biofunctionalized nanoslits in the LAAS cleanroom where the biosensor consists of a funtionalized gold layer embedded in a silicon nanochannel and chip encapsulation is carried out by means of a hard-PDMS covered cover slip.
- The grafting protocol for sensor functionalization was developed along with an adequate surface passivation strategy used to block the non-reactive surfaces of the nanochannels.
- An alternative bonding protocol using epoxy-silane modification of glass surfaces was proposed in order to allow the immobilization of proteins directly deposited in the nanoslit by means of a commercial spotter.
WP 2:
- A Finite Element Model of the nanoslit platform allowing the extraction of binding constants from experimental sigmoids was developed.
- A simplified one-dimensional model, taking into account our specific non-diffusion limited regime, was also implemented.
WP 3:
- Electrokinetic driven flow was used in nanochannel biosensor in order to improve the mass transport.
WP 4:
- Two representative protein-receptor pairs of different affinities, streptavidin-biotin (high affinity) and mouse IgG/anti-mouse IgG (medium affinity), were selected to demonstrate the capability of our devices to determine kinetic parameters.
- Kinetic experiments were conducted in biofunctionalized nanoslits using the selected biological models.
- Kinetic measurements were conducted using SPR on the IgG/anti-IgG pair.
- We have observed a good correlation between extracted association/dissociation constants and on/off rates measured with SPR and values reported in the literature, thus demonstrating the ability of the nanoslit biosensor proposed in this project for kinetic constant determination.

- Demonstration of the use of our nanoslit biosensor for kinetic studies: the extracted association and dissociation constants of two relevant binding pairs are in accordance with literature data and with SPR derived values
- Development of a nanoslit-embedded multiplexed immunosensor

1. T. Leïchlé, and C.-F. Chou, «Biofunctionalized nanoslits for wash-free and spatially resolved real-time sensing with full target capture«, Biomicrofluidics, 9, 034103 (2015)
2. P. Teerapanich, M. Pugniere, Y. L. Lin, C. F.Chou, and T. Leichle, «Biofunctionalized nanoslits for fluorescence monitoring of protein binding kinetics«, XII Conference on Optical Chemical Sensors and Biosensors (Europtrode 2014), Athens, Greece, April 13-16, 2014
3. P. Teerapanich, M. Pugniere, Y. L. Lin, C. F.Chou, and T. Leichle, «Determination of protein binding kinetics using a simple slit-like nanofluidic biosensor«, Biosensors 2014, Melbourne, Australia, May 27-30, 2014

Early diagnosis and screening of many diseases such as cancer, epidemic influenza, and myocardial infarction (MI), is widely recognized as to greatly increase successful treatment and improve the patient’s quality of life. In 2005, the 58th World Health Assembly approved a resolution urging its member states to give priority to research of early cancer detection. However, significant bottlenecks exist for effective detection at the early stage of a disease. One of the major challenges is the difficulty in detection at the low concentration of cancer biomarkers at the early stage. Traditional physical detection methods are limited by the low signal quality, while chemical detection methods are limited by the low reaction rate. To overcome this obstacle, novel technologies to filter and detect the specific biomarkers need to be developed and implemented. Given the fact that our Consortium members are the first in demonstrating: (1) the development of fabrication protocols for pre-biofunctionalized nanoslit sensors for real-time sensing with spatially resolved reaction kinetics, (2) surface modification scheme for orientated protein sensor immobilization, and (3) ultrafast protein enrichment, demonstrated 105-fold enrichment in 20 seconds (a world record speed), using the concept of molecular damming effect by nanoscale electrodeless dielectrophoresis. By integrating these methodologies developed within the Consortium, we aim to develop a novel integrated biofunctional nanofluidic system for real-time, ultrasensitive, spatially resolved and potentially multiplexed diagnosis for cancer and other disease biomarkers. The multi-disciplinary team assembled here includes expertise from biophysics, micro/nanofluidics, micro/nanofabrication, protein chemistry, surface functionalization, to surface plasmon resonance (SPR) sensors, which are organized to tackle this important challenge, using two model biomarkers: prostate cancer biomarker (prostate specific antigen, or PSA) and cardiovascular biomarker for MI, Troponin I. The outcome of this study should provide practical and low-cost solution to the health care industry and may be extended to general disease biomarker detection and thus benefit the human society in general.