Multicolor surface plasmon resonance imaging of ink jet-printed protein microarrays

We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules.     

Surface plasmon resonance phase imaging measurements of patterned monolayers and DNA adsorption onto microarrays

The optical technique of surface plasmon resonance phase imaging (SPR-PI) is implemented in a linear microarray format for real-time measurements of surface bioaffinity adsorption processes. SPR-PI measures the phase shift of p-polarized light incident at the SPR angle reflected from a gold thin film in an ATR Kretschmann geometry by creating an interference fringe image on the interface with a polarizer-quartz wedge depolarizer combination. The position of the fringe pattern in this image changes upon the adsorption of biomolecules to the gold thin film. By using a linear array of 500 μm biosensor element lines that are perpendicular to the interference fringe image, multiple bioaffinity adsorption measurements can be performed in real time. Two experiments were performed to characterize the sensitivity of the SPR-PI measurement technique: First, a ten line pattern of a self-assembled monolayer of 11-mercaptoundecamine (MUAM) was created via photopatterning to verify that multiple phase shifts could be measured simultaneously. A phase shift difference (Δφ) of Δφ = 182.08 ± 0.03° was observed for the 1.8 nm MUAM monolayer; this value agrees with the phase shift difference calculated from a combination of Fresnel equations and Jones matrices for the depolarizer. In a second demonstration experiment, the feasibility of SPR-PI for in situ bioaffinity adsorption measurements was confirmed by detecting the hybridization and adsorption of single stranded DNA (ssDNA) onto a six-component DNA line microarray patterned monolayer. Adsorption of a full DNA monolayer produced a phase shift difference of Δφ = 28.80 ± 0.03° at the SPR angle of incidence and the adsorption of the ssDNA was monitored in real time with the SPR-PI. These initial results suggest that SPR-PI should have a detection limit roughly 100 times lower than traditional intensity-based SPR imaging measurements.     

Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays

Surface plasmon resonance (SPR) imaging is a surface-sensitive spectroscopic technique for measuring interactions between unlabeled biological molecules with arrays of surface-bound species. In this paper, SPR imaging is used to quantitatively detect the hybridization adsorption of short (18-base) unlabeled DNA oligonucleotides at low concentration, as well as, for the first time, the hybridization adsorption of unlabeled RNA oligonucleotides and larger 16S ribosomal RNA (rRNA) isolated from the microbe Escherichia coli onto a DNA array. For the hybridization adsorption of both DNA and RNA oligonucleotides, a detection limit of 10 nM is reported; for large (1,500-base) 16S rRNA molecules, concentrations as low as 2 nM are detected. The covalent attachment of thiol-DNA probes to the gold surface leads to high surface probe density (10(12) molecules/cm2) and excellent probe stability that enables more than 25 cycles of hybridization and denaturing without loss in signal or specificity. Fresnel calculations are used to show that changes in percent reflectivity as measured by SPR imaging are linear with respect to surface coverage of adsorbed DNA oligonucleotides. Data from SPR imaging is used to construct a quantitative adsorption isotherm of the hybridization adsorption on a surface. DNA and RNA 18-mer oligonucleotide hybridization adsorption is found to follow a Langmuir isotherm with an adsorption coefficient of 1.8 x 10(7) M(-1).     

Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy

A simple method for converting local reflectivity changes measured in surface plasmon resonance (SPR) microscopy to effective adlayer thicknesses and absolute surface coverages of adsorbed species is presented. For a range of high-contrast angles near the SPR resonance where the local metal surface's reflectivity changes linearly with angle, the change in reflectivity at fixed angle is proportional to the change in effective refractive index (eta(eff)) near the surface. This change in eta(eff) can be converted to absolute adsorbate coverage using methods developed for quantitative SPR spectroscopy. A measurement of the change in reflectivity due to changes in refractive index of bulk solutions, i.e., percent reflectivity change per refractive index unit (RIU), is the only calibration required. Application of this method is demonstrated for protein adsorption onto protein/DNA arrays on gold from aqueous solution using an SPR microscope operating at 633 nm. A detection limit of 0.072% change in absolute reflectivity is found for simultaneous measurements of all 200 microm x 200 microm areas within the 24-mm(2) light beam with 1-s time averaging. This corresponds to a change in effective refractive index of 1.8 x 10(-5) and a detection limit for protein adsorption of 1.2 ng/cm(2) (approximately 0.5 pg in a 200-microm spot). The linear dynamic range is Deltaeta(eff) = approximately 0.011 RIU or approximately 720 ng/cm(2) of adsorbed protein. Using a nearby spot as a reference channel, one can correct for instrumental drift and changes in refractive index of the solutions in the flow cell.     

Near infrared surface plasmon resonance phase imaging and nanoparticle-enhanced surface plasmon resonance phase imaging for ultrasensitive protein and DNA biosensing with oligonucleotide and aptamer microarrays

The techniques of surface plasmon resonance-phase imaging (SPR-PI) and nanoparticle-enhanced SPR-PI have been implemented for the multiplexed bioaffinity detection of proteins and nucleic acids. The SPR-PI experiments utilized a near-infrared 860 nm light emitting diode (LED) light source and a wedge depolarizer to create a phase grating on a four-element single-stranded DNA (ssDNA) microarray; bioaffinity adsorption onto the various microarray elements was detected via multiplexed real time phase shift measurements. In a first set of demonstration experiments, an ssDNA aptamer microarray was used to directly detect thrombin at concentrations down to 100 pM with SPR-PI. Two different ssDNA aptamers were used in these experiments with two different Langmuir adsorption coefficients, K(A1) = 4.4 × 10(8) M(-1) and K(A2) = 1.2 × 10(8) M(-1). At concentrations below 1 nM, the equilibrium phase shifts observed upon thrombin adsorption vary linearly with concentration with a slope that is proportional to the appropriate Langmuir adsorption coefficient. The observed detection limit of 100 pM is approximately 20 times more sensitive than that observed previously with SPRI. In a second set of experiments, two short ssDNA oligonucleotides (38mers) were simultaneously detected at concentrations down to 25 fM using a three-sequence hybridization format that employed 120 nm DNA-modified silica nanoparticles to enhance the SPR-PI signal. In this first demonstration of nanoparticle-enhanced SPR-PI, the adsorbed silica nanoparticles provided a greatly enhanced phase shift upon bioaffinity adsorption due to a large increase in the real component of the interfacial refractive index from the adsorbed nanoparticle. As in the case of SPR-PI, the detection limit of 25 fM for nanoparticle-enhanced SPR-PI is approximately 20 times more sensitive than that observed previously with nanoparticle-enhanced SPRI.     

Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions

A sensitive method for the analysis of single nucleotide polymorphisms (SNPs) in genomic DNA that utilizes nanoparticle-enhanced surface plasmon resonance imaging (SPRI) measurements of surface enzymatic ligation reactions on DNA microarrays is demonstrated. SNP identification was achieved by using sequence-specific surface reactions of the enzyme Taq DNA ligase, and the presence of ligation products on the DNA microarray elements was detected using SPRI through the hybridization adsorption of complementary oligonucleotides attached to gold nanoparticles. The use of gold nanoparticles increases the sensitivity of the SPRI so that single bases in oligonucleotides can be successfully identified at a concentration of 1 pM. This sensitivity is amply sufficient for performing multiplexed SNP genotyping by using multiple PCR amplicons and should also allow for the direct detection and identification of SNP sequences from 1 pM unamplified genomic DNA samples with this array-based and label-free SPRI methodology. As a first example of SNP genotyping, three different human genomic DNA samples were screened for a possible point mutation in the BRCA1 gene that is associated with breast cancer.     

Surface plasmon resonance imaging measurements of antibody arrays for the multiplexed detection of low molecular weight protein biomarkers

This paper describes a simple methodology for the creation of high-density multiplexed antibody arrays on gold surfaces that can be used to detect low molecular weight protein biomarkers with surface plasmon resonance imaging (SPRI). A one-step carbonyldiimidazole (CDI) surface reaction was utilized to attach antibodies onto alkanethiol-modified gold surfaces and characterized with polarization modulation FT-IR reflection absorption spectroscopy. The CDI chemistry was then employed to create an antibody microarray with array element sizes varying from 750 microm down to 200 microm. As a demonstration, a three-component antibody array was employed to detect two clinically important protein biomarkers, beta2-microglobulin (11.8 kDa) and cystatin C (13.4 kDa). SPRI measurements could simultaneously detect both of these small unlabeled proteins with no cross talk at solution concentrations from 300 nM down to 1 nM. In addition, the adsorption strengths of these biomarkers onto an antibody array were measured with SPRI and compared to those obtained from the kinetic analysis of single-channel angle shift SPR measurements.     

Detection of protein biomarkers using RNA aptamer microarrays and enzymatically amplified surface plasmon resonance imaging

A methodology for the detection of protein biomarkers at picomolar concentrations that utilizes surface plasmon resonance imaging (SPRI) measurements of RNA aptamer microarrays is developed. The adsorption of proteins onto the RNA microarray is detected by the formation of a surface aptamer-protein-antibody complex. The SPRI response signal is then amplified using a localized precipitation reaction catalyzed by the enzyme horseradish peroxidase that is conjugated to the antibody. This enzymatically amplified SPRI methodology is first characterized by the detection of human thrombin at a concentration of 500 fM; the appropriate thrombin aptamer for the sandwich assay is identified from a microarray of three potential thrombin aptamer candidates. The SPRI method is then used to detect the protein vascular endothelial growth factor (VEGF) at a biologically relevant concentration of 1 pM. VEGF is a signaling protein that has been used as a serum biomarker for rheumatoid arthritis, breast cancer, lung cancer, and colorectal cancer and is also associated with age-related macular degeneration.     

Detection of electrochemical enzymatic reactions by surface plasmon resonance measurement

We describe the surface plasmon resonance (SPR) detection of an enzymatic turnover reaction and the measurement of glucose concentration using a multienzyme layer modified gold electrode. We constructed an osmium redox polymer mediated enzyme sensor on a gold thin-film electrode and monitored electrochemical reaction by SPR measurement. Unlike the usual binding assay with SPR, here we used SPR to detect the redox state of an electron mediator that was the result of the electron-transfer reaction of sequential enzymatic reactions. Therefore, the degree of refractive index change was independent of the dielectric property of the substrate and enzymatic molecular recognition was converted to refractive index change with amplification. For the quantitative evaluation of glucose with this method, we used chronopotentiometry and a linear relation was obtained between the glucose concentration and the rate of refractive index change.     

Determination of ribonuclease H surface enzyme kinetics by surface plasmon resonance imaging and surface plasmon fluorescence spectroscopy

The kinetics of the ribonuclease H (RNase H) surface hydrolysis of RNA-DNA heteroduplexes formed on DNA microarrays was studied using a combination of real-time surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS). Time-dependent SPRI and SPFS data at various enzyme concentrations were quantitatively analyzed using a simple model that couples diffusion, enzyme adsorption, and surface enzyme kinetics. This model is characterized by a set of three rate constants, enzyme adsorption (k(a)), enzyme desorption (k(d)), enzyme catalysis (k(cat)), and one dimensionless diffusion parameter (beta). Values of k(a) = 3.15 (+/-0.20) x 10(6) M(-1).s(-1), k(d) = 0.10 (+/-0.05) s(-1), and k(cat) = 0.95 (+/-0.10) s(-1) were determined from fitting all of the SPRI and SPFS data sets. One of the most interesting kinetic parameters is the surface RNase H hydrolysis reaction rate constant (k(cat)), which was found to be approximately 10 times slower than that observed in solution, but approximately 100 times faster than that recently observed for the exonuclease III surface hydrolysis of double-stranded DNA microarrays (k(cat) = 0.009 s(-1)). Moreover, the surface coverage of the intermediate enzyme-substrate complex (ES) was found to be extremely small during the course of the reaction because k(cat) is much larger than the product of k(a) and the bulk enzyme concentration.     

Etched glass microarrays with differential resonance for enhanced contrast and sensitivity of surface plasmon resonance imaging analysis

We report the fabrication and characterization of gold-coated etched glass array substrates for surface plasmon resonance imaging (SPRi) analysis with significantly enhanced performance, in particular image contrast and sensitivity. The etching of the glass substrate induces a variation in the resonance condition and thus in the resonance angle between the etched wells and the surrounding area, leading to the isolation of the array spot resonance with a significant reduction of the background signal. FDTD simulations show arrays with large spots and minimal spot-to-spot spacing yield ideal differential resonance conditions, which are verified by experimental results. Simulations also indicate the etched well structure exhibits enhanced SPR electric field intensity by 3-fold as compared to standard planar gold chips. Changes in the bulk sensitivity of the etched arrays have been obtained at the 10(-4) RIU level based on image intensity difference. The strong image contrast allows for improved microarray imaging analysis with easily distinguished signals from background resonance. The etched array chips are demonstrated for SPRi detection of bacterial toxins through the coating of an ultrathin SiO(2) film for direct vesicle fusion that establishes a supported membrane-based biosensing interface. Protein detection with cholera toxin (CT) at 5 nM is obtained, making this chip one of the most sensitive SPR imaging substrates ever reported without a postbinding amplification scheme. Furthermore, the surface can be regenerated by Triton X-100 for repeated cycles of membrane formation, protein binding, and biomolecular removal. The reusability and enhanced performance of the etched glass array chips should find a broad range of applications, opening up new avenues for high-throughput SPR imaging detection with convenience and marked surface sensitivity.     

Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging

The characterization of peptide arrays on gold surfaces designed for the study of peptide-antibody interactions using surface plasmon resonance (SPR) imaging is described. A two-step process was used to prepare the peptide arrays: (i) a set of parallel microchannels was used to deliver chemical reagents to covalently attach peptide probes to the surface by a thiol-disulfide exchange reaction; (ii) a second microchannel with a wraparound design was used as a small-volume flow cell (5 microL) to introduce antibody solutions to the peptide surface. As a demonstration, the interactions of the FLAG epitope tag and monoclonal anti-FLAG M2 were monitored by SPR imaging using a peptide array. This peptide-antibody pair was studied because of its importance as a means to purify fusion proteins. The surface coverage of the FLAG peptide was precisely controlled by creating the peptide arrays on mixed monolayers of alkanethiols containing an amine-terminated surface and an inert alkanethiol. The mole fraction of peptide epitopes was also controlled by reacting solutions containing FLAG peptide and the non-interacting peptide HA or cysteine. By studying variants based on the FLAG binding motif, it was possible to distinguish peptides differing by a single amino acid substitution using SPR imaging. In addition, quantitative analysis of the signal was accomplished using the peptide array to simultaneously determine the binding constants of the antibody-peptide interactions for four peptides. The binding constant, K(ads), for the FLAG peptide was measured and found to be 1.5 x 10(8) M(-1) while variants made by the substitution of alanine for residues based on the binding motif had binding constants of 2.8 x 10(7), 5.0 x 10(6), and 2.0 x 10(6) M(-1).     

Enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.     

Enzymatically amplified surface plasmon resonance imaging method using RNase H and RNA microarrays for the ultrasensitive detection of nucleic acids

A novel surface enzymatic amplification method that utilizes RNA microarrays in conjunction with the enzyme RNase H is developed for the ultrasensitve detection and analysis of target DNA molecules. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from RNA-DNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance imaging, multiple DNA targets can be detected at a concentration of 10 fM on a single chip. In addition, this method is utilized for the sequence-specific detection of the TSPY gene in both purified and unpurified PCR products. Finally, in a series of kinetics measurements, the initial rate of hydrolysis is shown to depend directly on the surface concentration of DNA-RNA heteroduplexes.     

Deep UV nano-microstructuring of substrates for surface plasmon resonance imaging

In this paper, we describe wafer-scale fabrication and characterization of plasmonic chips-containing different sizes and spacings of metallic micro- and nanoline structures-using deep UV lithography. Using a high dose (25 mJ cm( - 2)) and a proper lift-off process, feature sizes as small as 25 nm are obtained. Moreover, we study the dependence of surface plasmon resonance on the angle of incidence and wavelength for different micro- and nanoline size and spacing values, yielding localized to quasi-propagative plasmonic behaviors. Rigorous coupled wave analysis (RCWA) techniques are employed to numerically confirm these experimental observations. Finally, the refractive index of media around the SPRI sensor chips is varied, showing the angulo-spectral regions of higher sensitivity for each type of structure.     

Real-time quantification of microscale bioadhesion events in situ using imaging surface plasmon resonance (iSPR)

From macro- to nanoscales, adhesion phenomena are all-pervasive in nature yet remain poorly understood. In recent years, studies of biological adhesion mechanisms, terrestrial and marine, have provided inspiration for "biomimetic" adhesion strategies and important insights for the development of fouling-resistant materials. Although the focus of most contemporary bioadhesion research is on large organisms such as marine mussels, insects and geckos, adhesion events on the micro/nanoscale are critical to our understanding of important underlying mechanisms. Observing and quantifying adhesion at this scale is particularly relevant for the development of biomedical implants and in the prevention of marine biofouling. However, such characterization has so far been restricted by insufficient quantities of material for biochemical analysis and the limitations of contemporary imaging techniques. Here, we introduce a recently developed optical method that allows precise determination of adhesive deposition by microscale organisms in situ and in real time; a capability not before demonstrated. In this extended study we used the cypris larvae of barnacles and a combination of conventional and imaging surface plasmon resonance techniques to observe and quantify adhesive deposition onto a range of model surfaces (CH(3)-, COOH-, NH(3)-, and mPEG-terminated SAMs and a PEGMA/HEMA hydrogel). We then correlated this deposition to passive adsorption of a putatively adhesive protein from barnacles. In this way, we were able to rank surfaces in order of effectiveness for preventing barnacle cyprid exploration and demonstrate the importance of observing the natural process of adhesion, rather than predicting surface effects from a model system. As well as contributing fundamentally to the knowledge on the adhesion and adhesives of barnacle larvae, a potential target for future biomimetic glues, this method also provides a versatile technique for laboratory testing of fouling-resistant chemistries.     

Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions

The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format.     

Real-time and full-field detection of near-wall salinity using surface plasmon resonance reflectance

An idea of real-time and full-field detection of near-wall salinity is presented to use the surface plasmon resonance (SPR) reflectance that changes with refractive index variations of the tested saline fluid. The laboratory-designed SPR system, based on the Kretschmann's configuration, uses a 47.5 nm thick gold layer as the SPR resonator, coated on a BK7 prism (n=1.515), and requires a one-time system calibration to establish a correlation of the specified saline mass concentration levels to the corresponding CCD (charge-coupled device) pixel gray levels. As a gravity-falling saline drop in water reaches the bottom and diffuses thereafter, the SPR system quantitatively maps the evolution of the salinity distributions in the near-wall region (less than 1 microm). An elaborate uncertainty analysis shows that the overall measurement uncertainties critically depend on the uniformity of the metal film thickness and the accuracy of its dielectric constant.     

Ultrasensitive microarray detection of short RNA sequences with enzymatically modified nanoparticles and surface plasmon resonance imaging measurements

A novel multiplexed method for short RNA detection that employs an enzymatic capture reaction onto DNA-modified silica nanoparticles (SiNPs) followed by nanoparticle-enhanced surface plasmon resonance imaging (SPRI) is demonstrated. SiNPs functionalized with 5'-phosphorylated single stranded DNA (ssDNA) are used with T4 RNA ligase to capture various short 20-24 base single-stranded RNA (ssRNA) oligonucleotides from a target solution. The ssRNA-modified SiNPs are collected from the target solution, specifically adsorbed onto a cDNA microarray and then detected with SPRI. The use of DNA-modified SiNPs to capture ssRNA for profiling has several advantages as compared to a planar SPRI surface bioaffinity adsorption format: (i) the target solution is exposed to a larger total surface area for the RNA ligation reaction; (ii) the SiNPs enhance the diffusion rate of the ssRNA to the surface; (iii) the SiNPs can be collected, washed, and preconcentrated prior to detection; and (iv) the ssRNA-modified SiNPs give an enhanced SPRI signal upon hybridization adsorption to the microarray. Our initial measurements demonstrate that this detection method can be used to detect multiple ssRNA sequences at concentrations as low as 100 fM in 500 μL.     

Integrated label-free protein detection and separation in real time using confined surface plasmon resonance imaging

We demonstrate a label-free protein detection and separation technology for real-time monitoring of proteins in micro/nanofluidic channels, confined surface plasmon resonance imaging (confined-SPRi). This was achieved by fabricating ultrathin fluidic channels (500 nm high, 500 microm wide) directly on top of a specialized SPRi sensor surface. In this way, SPRi is uniquely used to detect proteins deep into the fluidic channel while maintaining high lateral accuracy of separated products. The channel fluid and proteins were driven electrokinetically under an external electric field. For this to occur, the metallic SPR sensor (46 nm of Au on 2 nm of Cr) was segmented into an array of squares (each 200 microm x 200 microm in size and spaced 8 microm apart) and coated with 30 nm of CYTOP polymer. In this work, we track label-free protein separation in real time through a simple cross-junction fluidic device with an 8-mm separation channel length under 30 V/cm electric field strength.