A CMOS Multi-Functional Biosensor Array for Rapid Low-Concentration Analyte Detection with On-Chip DEP-Assisted Active Enrichment and Manipulation with No External Electrodes

Electronic biosensors are transducers that convert biomolecular or cellular information to electronic readouts. Though advances in electronics keep improving biosensors' performance, often their ultimate detection limits and functionalities are constrained at the sensor interfaces. For instance, existing electrochemical or optical bioanalytical platforms commonly face challenges in immobilizing surface bioreceptors and detecting ultra-trace biospecimens due to the slow mass transport limitation from the bulk solution. Additional target amplification or complex signal enhancement are thus needed. Therefore, there is a perennial need to explore biology-electronics co-designs to overcome some of these interface limitations and further advance biosensor detection capabilities.

Electronic biosensors are transducers that convert biomolecular or cellular information to electronic readouts. Though advances in electronics keep improving biosensors' performance, often their ultimate detection limits and functionalities are constrained at the sensor interfaces. For instance, existing electrochemical or optical bioanalytical platforms commonly face challenges in immobilizing surface bioreceptors and detecting ultra-trace biospecimens due to the slow mass transport limitation from the bulk solution. Additional target amplification or complex signal enhancement are thus needed. Therefore, there is a perennial need to explore biology-electronics co-designs to overcome some of these interface limitations and further advance biosensor detection capabilities.
One attractive molecular/cellular manipulation technique is dielectrophoresis (DEP) that applies active analyte transport to address interface limitations. Some early CMOS sensor platforms explore DEP on-chip [1][2][3]. However, they often require external electrodes or cover electrodes, such as ITO electrodes to drive DEP, making the chip packaging difficult and unreliable. Moreover, these reported CMOS DEP chips only contain impedance sensing modality, which limits their functionalities. We propose a DEP-assisted CMOS multi-functional molecular sensor platform for enhanced analyte detection without external electrodes or conductive covers. The on-chip DEP performs manipulation and enrichment of analytes, ensuring fast and reliable low-concentration detections, ideal for rapid sample-in-answer-out environmental monitoring and point-of-care (PoC) devices. Each sensing pixel contains independently controllable and functional three photodiodes, and one in-pixel working electrode (WE) shared by DEP, impedance mapping, and electrochemical modalities and DEP actively transport target analytes to the interface for enhanced detections (Fig. 21.3.1). First, the EC sensors and DEP together achieve higher limit-of-detection (LoD). Since on-chip DEP achieves active transport and analyte enrichment, the EC sensors thus can detect the low concentration analyte. Also, thanks to the DEP-based enrichment, the impedance and EC sensors achieve rapid analysis. Lastly, the DEP-based particle manipulations is readily detected by on-chip optical sensor arrays to ultimately achieve close-loop manipulation and sensing, such as droplet microfluidics.
The physical principle of DEP-based active transport is introduced in Fig. 21.3.1 [4]. An external actuation electrical field will polarize the particles/samples. Strong electrical field and particles with high polarizability make the DEP potential energy magnitude higher and facilitate particle trapping and controlling. If DEP potential energy magnitude is higher than its thermal energy, particles can be manipulated by DEP. However, if the thermal energy is higher than the DEP potential energy magnitude, the Brownian motion is dominant, rendering the DEP manipulation less effective. To yield strong polarization electrical field, our proposed CMOS sensor platform applies differential AC actuation voltages across two adjacent working electrodes (WEs), while the spacing of WEs are carefully designed to ensure sufficient DEP polarization. Note the on-chip DEP reuses the same WEs for electrochemical potentiostats and impedance mapping for area saving, and no external electrode is needed.
Our CMOS biosensor platform contains 4096 WEs, each of 28μm × 28μm. The edge-toedge spacing between WEs is 22μm. The relative permittivity and conductivity of water are used in simulations. Also, the temperature and polarizability of particles are set up as 298K and 50 x 10 -31 Fm 2 . It should be mentioned that media with low-ionic concentrations enhance DEP polarization, while many molecular and bacteria sensing can be performed in such media and oil-droplet based microfluidics [5,6]. Our platform uses CMOS-compatible 3V p-p swing with opposite phases on each WE for DEP polarization. The simulation result shows that our design is capable of trapping and manipulating analytes for active transport and enrichment up-to 37μm above the WEs.
Our DEP-assisted CMOS sensor array chip is shown in Fig. 21.3.2. The array chip contains four pixel groups. Each pixel group is made of 1024 28μm x 28μm WEs, 320 28μm x 28μm counter electrodes (CE), 64 28μm x 28μm reference electrodes (RE), and 3072 photodiodes (PD). Also, each pixel group has a programmable DEP controller, an impedance sensing block, an EC potentiostat, 4 optical detection blocks, and 4 programmable output stages. The impedance sensing block makes excitation signals in five frequency levels from 15kHz to 500kHz. The internal digital switch logic and PDreset logic are also integrated.
To demonstrate the DEP-assisted multi-functional analyte sensing of our CMOS biosensor array, we perform the following biosensing experiments.
We first utilize the resazurin (RS) redox process as a biomarker to detect the presence of living E. coli bacteria for rapid environment monitoring and food safety screening (Fig.  21.3.3). The reduction of RS is due to the loss of an oxygen atom loosely bound to the nitrogen atom of the phenoxazine nucleus. The reduction process follows two steps that are observed by cyclic voltammetry (CV) using on-chip potentiostat sensing modality. Firstly, the weakly fluorescent RS is irreversibly reduced into resorufin by the presence of the living E. coli bacteria. This step generates the main reduction peak current in CV. The resorufin can be further reduced into dihydroresorufin, observable as the second weaker reduction current peak. This second step is also reversible by atmospheric oxygen. Without E. coli, the reduction peak current of RS stays constant over time. In comparison, a clear and time-dependent decrease of amperometric peak current indicates the irreversible reduction of RS caused by living E. coli. This enables the rapid detection of living E. coli bacteria using our on-chip EC sensors. We load 2 x 10 7 copies/ml E. coli with 250μM RS onto our CMOS sensor and conduct CV measurement over 40 minutes. The CV readout shows that our platform can clearly detect the reduction current of RS. Also, CA measurements in Fig. 21.3.3 show the reduction peak current continuously decreases over time, demonstrating rapid living E. coli detection by the on-chip EC potentiostat in our CMOS sensor array.
Many biosensing applications require detections of low-concentration targets/analytes and high dynamic range. An example is water quality monitoring when E. coli bacteria concentration is low in clean drinkable water. In this case, most sensing platforms cannot yield conclusive results without long sensing time, e.g., for the E. coli to diffuse to the sensor surface or for E. coli proliferation. These LoD sensor issues can be solved by DEP-based analyte enrichment (Fig. 21.3.4). We label E. coli with 2μm-diameter anti-E. coli antibodies modified microbeads to show the DEP-based enrichment and the rapid detection. The DEP force acts on the microbeads that actively concentrate E.coli bacteria onto WEs and enhance the detection signal. In the experiment, 1kHz DEP actuation voltage is applied to the target electrode array. The measured time-lapse image of DEPbased enrichment is shown. The microbeads are rapidly enriched in the DEP-applied region. The CV scanning is conducted using the WEs both inside and outside of the DEPapplied region. The CV readout shows that the reduction peak current of the DEP-applied region decreases faster than that outside of DEP region. This demonstrates that our DEPassisted biosensor platform can radically improve the detection speed and LoD (also dynamic-range) for low-concentration analytes. Figure 21.3.5 shows the joint operation of DEP with the PD optical detection and impedance sensor. First, we demonstrate on-chip sample manipulation by reprogrammable DEP that is further monitored by on-chip PD sensor array. 100μmdiameter polystyrene microbeads are moved in X-/Y-directions by reprogrammable on-chip DEP forces. The bead movements are accurately tracked by PD arrays, which well match the reference microscopic images. Also, for the impedance sensing with DEP, DEP enrichment and impedance mapping are performed both at 15kHz. The 2D impedance change and the microscopic images show that enriched E. coli and microbeads are accurately detected by the impedance sensing block. These on-chip optical and impedance detections jointly with on-chip programmable DEP manipulation shows the capabilities of self-contained DEP analyte manipulation with multi-functional biosensing, useful for applications like automated large-scale tissue engineering and cell manufacturing.