On-chip microscale isoelectric focusing enhances protein detection limit

Abstract

Enhancing the detection limit in protein analysis is essential for a wide range of biomedical applications. In typical fluorescent protein assays, this limit is constrained by the detection capacity of the photon detector. Here, we develop an approach that significantly enhances the protein detection threshold by using microscale isoelectric focusing implemented directly at the detection site on a protein sensor chip. We demonstrate that by electrically generating a localized pH environment within a radius of ∼60 μm, protein molecules can be concentrated within this range and be detected at levels over four times lower than those achieved by measurements without on-chip isoelectric focusing. We find that this detection-limit enhancement results from a dual effect: the concentrating of the protein molecules and a reduction in the diffusion-induced fluctuation. Our approach offers a simple, yet highly effective ultra-low-power all-electronic solution for substantially improving protein analysis detection limits for diverse applications, including healthcare, clinical diagnostics, and therapeutics.

High-sensitivity quantification of proteins is pivotal for advancements in various biomedical applications including diagnostics and therapeutics.^1–5 Identifying weak fluorescent signals against background noise is key in detecting ultra-low amount of fluorescent protein. However, the detection limit of typical fluorescent protein assays is constrained by the photon detector.⁶ Typical methods for improving detection limits have primarily focused on optimizing the design of fluorophores,^7,8 improving photon detection apparatus,⁹ and developing fluorescence enhancement substrates.^10,11 Isoelectric focusing, an electrokinetic-based method, has shown considerable promise in processing proteins in microfluidic devices.^12–19 Here, we develop an on-chip, all-electronic method using microscale isoelectric focusing (μIEF) to enhance the detection limit of proteins. By using a pair of concentric ring microelectrodes, as shown in Fig. 1(a), a localized pH environment can be formed within 20 s in the region enclosed by the inner ring microelectrode within a radius of ∼60 μm, at a power consumption of <0.1 μW. The protein molecules can be concentrated within this range and detected at the concentration spot. This μIEF method, fully compatible and easily integrable with existing fluorescent techniques, enhances the detection limit for protein molecules by more than fourfold compared to the measurements that do not use μIEF.

FIG. 1.

The focusing and sensing of proteins upon pH-gradient generation between two concentric ring microelectrodes. (a) Schematic of microscale isoelectric focusing (μIEF). Protein molecules are represented by orange particles. (b) Optical image of the device. The scale bar is 1 mm. (c) Microscopic image for the generated pH gradient indicated by phenol red. The scale bar is 50 μm.

The device [Fig. 1(b)] for implementing μIEF is fabricated using scalable methods (see supplementary material methods—device fabrication section and Fig. S1 for details). The fluidic channel is 143 μm in thickness and 500 μm in width. In the fluidic channel, a pair of concentric ring gold microelectrodes with a thickness of 60 nm is exposed to solution with other areas passivated by a layer of photoresist.

To generate the pH gradient, a 25-nA current bias is applied between the microelectrodes (see supplementary material methods—pH-gradient generation section for details). The pH gradient is formed within 20 s after applying the current bias and remains stable in the fluidic channel for over 10 min under a flowless condition, accompanied by a stabilized voltage of 2.09 ± 0.01 V. The stable pH gradient creates a localized environment of acidity around the inner electrode, spanning an area with a diameter of approximately 60 μm, as shown in Fig. 1(c).

The pH gradient concentrates protein in the solution, specifically bovine serum albumin (BSA) tagged by Alexa Fluor 555 in our experiment,²⁰ through μIEF, as shown by the video in the supplementary material. Figure 2(a) shows the distribution profile of the μIEF-concentrated protein molecules along the x-axis. The radial fluorescence intensity profiles at various heights, interrogated by confocal fluorescence microscopy and shown in Fig. 2(b), indicate that the thickness of the molecule layer concentrated by μIEF, corresponding to that of the localized acidity, is approximately 40 μm (see supplementary material methods—confocal Z-stacking imaging section for details). Hence, the tagged BSA molecules are concentrated within a volume of ∼500 pl.

FIG. 2.

Fluorescence detection of the μIEF-concentrated BSA molecules. (a) Fluorescence microscopic image. The curve shows the emission intensity (AU) along the dash line. The scale bar is 50 μm. (b) Confocal radial fluorescence intensity profiles at different heights. (c) Fluorescence emission from the tagged BSA molecules within the range enclosed by the inner microelectrode vs the bulk BSA concentration. The lines are fitted using a linear function for data points that are above the background noise level. The horizontal line is used for eye-guiding. The error bars represent the standard deviation of multiple measurements.

The μIEF of the protein molecules significantly improves their detection limit. We measure the emission intensity of the tagged BSA molecules at various concentrations, both using μIEF and under conditions without μIEF (see supplementary material methods—BSA sensing section for details). As illustrated in Fig. 2(c), the emission intensity of BSA without using μIEF, when compared to the buffer, remains approximately zero for concentrations up to 25 pM. This is attributed to the limitations of the photon detector. For BSA concentrations above 25 pM, the data represent a clear linear relationship between the emission intensity and the concentration. The error bars in the data are due to the fluctuations of the fluorescent light intensity caused by the diffusion of molecules and the particle-number fluctuation within the observation volume.²¹ The linear fitting to the data leads to a sensitivity (slope) of 0.20 ± 0.01 AU pM⁻¹ and an x-intercept of 36 ± 2 pM. The detection limit, defined as the concentration at which the mean fluorescence intensity of the sample exceeds that of the pure buffer solution by threefold of the buffer's fluorescence intensity error, is 61 pM.

Linear fitting to the data from the experiment based on μIEF yields a sensitivity of 0.19 ± 0.01 AU pM⁻¹, in excellent agreement with the sensitivity obtained under conditions where μIEF is not used. The x-intercept is 8 ± 2 pM, representing a significant leftward shift by 27 ± 4 pM in comparison with the μIEF-free measurement. The magnitude of the leftward shift can be elucidated through thermodynamic analysis. At equilibrium, the electrochemical potential for a BSA molecule in bulk solution, ${\mu }_{\mathrm{o}}+{\epsilon }_{\mathrm{o}}$, where ${\mu }_{\mathrm{o}}$ is the chemical potential in bulk solution and ${\epsilon }_{\mathrm{o}}$ is the electrostatic energy density, is equal to ${\mu }_{\mathrm{i}}$, the BSA chemical potential in the concentrated range. Therefore, at the low concentration range for BSA as in our experiment, the difference in concentration between the concentrated range and the bulk, $\Delta \left[C\right]$, is equal to ${\epsilon }_{\mathrm{o}}/RT$, where $R$ is the ideal-gas constant and $T$ is the temperature (∼300 K). In 20 s, the energy-density input induced by 25 nA of current through the buffer with a resistivity of about 20 Ω m into a range of a half sphere with a radius of ∼40 μm is approximately 7.5 × 10⁻⁵ J m⁻³.²² Therefore, $\Delta \left[C\right]$ is approximately 30 pM, in good agreement with that of the μIEF-induced leftward shift (27 ± 4 pM).

Moreover, the μIEF-involved method delivers a ∼2.8 fold lower mean standard deviation, 0.59 ± 0.16 AU, compared to that (1.64 ± 0.06 AU) based on the experiment without using μIEF. This is due to the reduction of the diffusion of molecules and the particle number fluctuations by the concentrating effect. The combination of the leftward shift of the response and the reduction of the noise, both induced by μIEF, delivers a detection limit of 15 pM, an over fourfold enhancement over the measurements that do not use μIEF.

In conclusion, we demonstrate an all-electronic microscale isoelectric focusing method for significantly improving the detection limit in fluorescent protein assays. Our approach features rapid processing time, minimal sample volume requirements, ultra-low energy consumption, and ease of integration with existing methods. These attributes offer considerable potential for advancements in various fields including research, healthcare, and diagnostics.

See the supplementary material for additional information on the methods, a figure for device fabrication process, and a video for μIEF.

AUTHOR DECLARATIONS

Conflict of Interest

The authors have no conflicts to disclose.

Author Contributions

Xiao Fan: Conceptualization (lead); Formal analysis (lead); Investigation (lead); Writing – original draft (lead). Xiaoyu Zhang: Investigation (supporting); Writing – review & editing (supporting). Huilu Bao: Investigation (supporting); Writing – review & editing (supporting). Xin Zhang: Investigation (supporting); Writing – review & editing (supporting). Jinglei Ping: Conceptualization (lead); Formal analysis (lead); Funding acquisition (lead); Supervision (lead); Writing – review & editing (lead).

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request.