Reference electrode design for ultra-low volume on-chip biochemical sensing

The reference electrode design developed by Saurabh Tomar and colleagues enables ultra-low volume biochemical sensing.

This research summary is based on the “Integration of Ultra-Low Volume Pneumatic Microfluidics with a Three-Dimensional Electrode Network for On-Chip Biochemical Sensing” research paper, authored by Saurabh Tomar, Charlotte Lasne, Sylvain Barraud, Thomas Ernst and Carlotta Guiducci. 

The paper was published in June 2021 in Micromachine Journal’s special “20 years of SU8 as MEMS Material” issue.

Abstract

The work describes a novel miniaturized pseudo reference electrode (RE) for ultra low volume biochemical sensing. The RE was designed for biasing Ion Sensitive Field Effect Transistors (ISFETs), eliminating the need for post-CMOS processing. The design can potentially scale up in numbers with the CMOS scaling and employs silane-mediated transfer of patterned gold electrode lines onto PDMS microfluidics, coating the inside of the microfluidic channel. “Through-PDMS-vias” (TPV) provide access to this electrode network. The TPV consists of high metal-coated SU-8 pillars and is manufactured by a novel process employing a patterned SU-8 adhesion depressor positive resist layer. The TPV and pseudo-RE networks were able to bias 1.5 nanoliters (nL) of isolated electrolyte volumes when integrated with pneumatic valves.

Introduction | Biochemical sensing and microfluidics 

Ion Sensitive Field Effect Transistors (ISFETs) were scaled down to 10 nm wide [1] and scaled up to hundreds of millions of transistors per chip [2]. Much research effort has been made to develop the silicon part of biosensing. Still, the microfluidic system has relatively lagged behind in research and development. 

ISFET biosensing makes use of big electrolyte pools with macroscopic reference electrodes for biochemical sensing, thereby not utilizing their full potential. To harness its full potential would be to be able to simultaneously perform several – from hundreds to thousands – of biosensing reactions in micro-sized biochemical incubation chambers that are mutually and electrolytically isolated. Each of these chambers  is monitored in real-time by one or more ISFET sensors. Such an approach requires parcellization strategies in microfluidics that can integrate a miniaturized reference electrode. 

Here, we propose a microfluidic design that addresses the above issues, all the while avoiding any post-CMOS processing.

 Aims

In short, this paper is about:

• fabrication of a novel reference electrode design and integrating it into microfluidics.
• processing steps to manufacture very high SU-8 structures with a high degree of surface cleanliness.
• demonstrating low-volume biosensing via ISFETs.

Setup and Results | Fabrication of the microfluidic module

Figure 1. (Left) 3D schematic of the microfluidic module on a CMOS chip showing the metal-coated “through-PDMS-vias”. (Right) The bottom view of the microfluidic module shows the layout of the gold electrode inside the microchannel.

EXPERIMENT SETUP | Designing the microfluidic module for biochemical sensing

The schematic design of the microfluidic module is depicted in Figure. 1. The microfluidic module consists of two PDMS layers, with the first thin layer housing the microfluidic channel and the gold reference electrode conformally integrated into it. A thicker second PDMS layer on the top of the first houses the pneumatic control channels and holes to allow electrical access to the reference electrode via the TPV for biochemical sensing.

Fabrication the device

The whole flow process was carried out on a 4-inch silicon wafer in the cleanroom. The master mold for pneumatic valves was created by reactive ion etching of silicon wafer. The process steps for the fabrication of the master mold of flow layer is shown in Figure 2. 

The fabrication starts with manufacture of mold of the microfluidic channel by standard photolithography. After passivation of the mold by evaporation of metal stack the gold electrodes were fabricated by sputtering a thin layer of gold, defining the electrode network by standard photolithography and etching with ion beam milling. Then 1mm high SU-8 pillars were fabricated by three consecutive coatings and exposure steps, followed by a single development step at the end. Thereafter, TPVs are realized by metalizing the SU-8 pillars by gold sputtering through a physical mask. 

The wafer was then silanized to enhance the gold electrode’s adhesion to PDMS. After protecting the TPV’s top with tape, a thin PDMS layer was spin-coated and partially cured. Meanwhile, a thick PDMS layer was drop casted over the pneumatic valve’s master mold. 

After dicing and demolding, the pneumatic PDMS layer was manually aligned with the thin PDMS layer of the flow channel and bonded by fully curing at 80°C for 2 hours. Finally, the microfluidic module was released from the master mold by sacrificial etching of the metal stack. 

SU-8 processing

The study reports a method for the fabrication of very high SU-8 pillars. Multiple spin coating of SU-8 are used to achieve the required height. The SU-8 layers are exposed after each softbake step. Multiple baking steps and long baking times partially polymerize a thin SU-8 layer in contact with the wafer (Figure 3, left). 

In applications like ours, where surface cleanliness after SU-8 development is critical, the residual SU-8 layer can be catastrophic to further processing. This issue can be mitigated by using a thin layer of positive resist prior to SU-8 coating as an adhesion de-promoter. 

The thin layer of positive resist prevents partially cross-linked SU-8 from adhering to wafer, thus guaranteeing a clean surface upon development (Figure 3, right). 

Electrical characterization

The PDMS microfluidic module was bonded to the CMOS chip via thermal bonding. The characterization was performed in the PM8 probe station. Electrolyte injection and actuation of

The stability of our reference electrodes was evaluated over time by measuring its open circuit potential (OCP) with respect to a commercial flow through Ag/AgCl electrode in a 3M KCl solution. The observed mean OCP drift of 2 mV/h is similar to other reported gold-based miniaturized reference electrodes.

The nanowire ISFET was gated via an isolated nanoliter-sized electrolyte volume, achieved by closing the ends of a 1 mm long section of the microchannel by the actuation of pneumatic valves after injection of KCl electrolyte. 

Thereafter ISFET was biased via the conformal gold electrode to demonstrate typical I-V characteristics (Figure 4). Under bias from the conformal electrode, the ISFET demonstrated an excellent static response (ID drift = ~1 nA/h) (Figure 5).

Conclusion | Biochemical sensing

Our new process for pseudo reference electrode (RE) integration in microfluidics, when combined with parcellization microfluidics, allows isolation and characterization of considerably small analyte volumes by ISFET. 

Small reaction volumes benefit from increased sensitivity, fewer contaminations and reduced cost. The design synergizes the advantages of low reaction volumes, unprecedented pH resolution, and CMOS scalability of ISFETs.

The approach is most suitable for label-free, semiconductor-based molecular diagnostic applications that require sample digitization.

References