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A Sample-to-Targeted Gene Analysis Biochip for Nanofluidic Manipulation of Solid-Phase Circulating Tumor Nucleic Acid Amplification in Liquid Biopsies Kevin Maisheng Koo, Shuvashis Dey, and Matt Trau ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01011 • Publication Date (Web): 21 Nov 2018 Downloaded from http://pubs.acs.org on November 24, 2018

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A Sample-to-Targeted Gene Analysis Biochip for Nanofluidic Manipulation of Solid-Phase Circulating Tumor Nucleic Acid Amplification in Liquid Biopsies

Kevin M. Koo,ǂ,¥ Shuvashis Dey,ǂ,¥ and Matt Trau*,ǂ,†

ǂCentre

for Personalized Nanomedicine, Australian Institute for Bioengineering and

Nanotechnology (AIBN), The University of Queensland, QLD 4072, Australia

†School

of Chemistry and Molecular Biosciences, The University of Queensland, QLD 4072,

Australia

¥Authors

contributed equally

Email: [email protected] (MT)

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ABSTRACT The use of circulating tumor nucleic acids (ctNA) in patient liquid biopsies for targeted genetic analysis is rapidly increasing in clinical oncology. Still, the call for an integrated methodology that is both rapid and sensitive for analyzing trace ctNA amount in liquid biopsies, has unfortunately not been fully realized. Herein, we performed complex liquid biopsy sample-totargeted genetic analysis on a biochip with 50 copies-detection limit within 30 min. Our biochip uniquely integrated: 1) electrical lysis and release of cellular targets with minimal processing; 2) nanofluidic manipulation to accelerate molecular kinetics of solid-phase isothermal amplification; 3) single-step capture and amplification of multiple NA targets prior to nanozyme-mediated electrochemical detection. Using prostate cancer liquid biopsies, we successfully demonstrated multifunctionality for cancer risk prediction; correlation of serum and urine analyses; and cancer relapse monitoring.

Keywords: microfluidics; solid-phase amplification; risk stratification; prostate cancer; liquid biopsy


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The field of liquid biopsy is currently in an exciting translational period in which the potential of targeted gene analysis without invasive biopsy procedures, is beginning to be demonstrated clinically for improved cancer management.1-3 Liquid biopsies involve the non-invasive sampling of oncogenic material such as circulating tumor nucleic acid (ctNA) biomarkers in blood or other biological fluids.4 Importantly, gene mutations that drive tumor growth are often present in different parts of the tumor, and liquid biopsies may hold a distinct crucial advantage in providing a “complete” sampling of mutations over tissue biopsy. Despite the clinical potential of liquid biopsies in cancer profiling, there is presently an unmet need for rapid yet accurate clinical targeted genetic analysis in liquid biopsies. The most common tools for enabling liquid biopsy applications are next-generation sequencing (NGS) and polymerase chain reaction (PCR) techniques. NGS provides sensitive and comprehensive genetic analysis5,6 but current cost and timeframe (~2 weeks) is still inhibitive for routine clinical use. In comparison, PCR-based techniques are quicker (~3 hours) and sensitive7,8 but require multi-step technical handling, as well as rigorous primer design to minimize multiplex amplification inaccuracies. Hence, it is favourable to devise a strategy which is fast (≤15 min), operator-friendly, and readily scalable for accurate analysis of multiple genes. Solid-phase NA amplification is a feasible technique for high-throughput analysis of multiple targets because of its ability to spatially encode primer arrays onto a surface. Particularly, this has allowed ideal incorporation of solid-phase amplification into chip-based technologies to develop automated sample-to-answer clinical biosensors that are suited for liquid biopsy applications.9,10 Yet, it is well-known that solid-phase amplification remains an inefficient process with low amplicon yields due to slow reaction kinetics on surfaceimmobilized primers.11 To realize the use of solid-phase amplification for rapid, cost-effective and concurrent multiple target analysis; we envision that chip-based nanofluidics could be a novel approach to enhance solid-phase amplification by increasing molecular collision frequency for greater amplification efficiency. Our laboratory has conceived a robust way of ACS Paragon Plus Environment

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generating electrohydrodynamics fluidic flow on electrode surfaces,12 and have recently applied it to enhance solution-based molecular kinetics13 for amplification-free NA biosensing. Due to its amplification-free detection nature, the required input sample amount still inhibits the bioassay from being widely applied for broad liquid biopsy sampling. In light of this limitation, we hereby remodeled our fluidic phenomenon to refine general liquid biopsy analysis by enhancing surface-confined (instead of solution-phase) reactions. Herein, we report a biochip system that performs an entire workflow of integrated ctNA target analysis (i.e. sample preparation, target amplification, and detection readout) from liquid biopsies. The core radical feature of our biochip lies in the first-time use of customized electrode patterning to create physical fluidic enhancement of solid-phase target amplification, thus achieving a detectable signal within an unprecedented 10 min amplification time. Moreover, our integrated biochip readily enables electrical release of cellular contents for rapid single-step target capture and isothermal amplification, and uses superparamagnetic iron oxide particles as stable

non-biological

peroxidase-mimicking

nanozymes14

during

electrochemical

measurements. Using prostate cancer (PC) as a model for liquid biopsy analysis, we demonstrated simultaneous targeted analysis of multiple PC genetic aberrations (including gene fusion and overexpression mutations)15-17 in patient samples. Furthermore, we investigated the biochip’s multifunctional clinical potential in predicting PC risk, studying biomarker correlation between serum and urine liquid biopsies within individual patients, and treatment monitoring after prostatectomy.


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EXPERIMENTAL SECTION Materials All reagents (Sigma Aldrich) were of analytical grade and used without further purification unless otherwise stated. UltraPureTM DNase/RNase-free distilled water (Invitrogen) was used throughout the experiments. Primer sequences (Integrated DNA Technologies) used in this work are shown in Table S1.

Biochip Design and Fabrication A square biochip layout design (Figure S1) containing a central sample lysis chamber and four individual target amplification/chronoamperometric detection chambers was prepared using a layout design software (L-edit MEMS-pro, v15). The lysis chamber comprised of a circular electrode (2000 µm diameter) surrounded by an outer ring (500 µm-thick) electrode with 500 µm separation (edge to edge). Each amplification/chronoamperometric detection chamber contained a pair of inner circular (800 µm diameter) and an outer ring (50 µm-thick) electrodes with 50 µm separation (edge to edge). All electrodes were individually connected to corresponding connection pads for supplying the biochip with external current input. The biochip design was then printed on a 5 inch-chrome mask using direct laser writer and developed by following a standard mask development procedure. Biochip fabrication was performed at Australian National Fabrication FacilityQueensland Node (ANFF-Q). The prepared mask was loaded in a mask aligner (EV Group) and UV-exposed to transfer the design onto AZnLof 2070 negative photoresist (MicroChem) coated (200 nm) silicon wafers. Post-exposure, the wafers were then developed using PGMEA (propylene glycol methyl ether acetate) developing solution, dried and loaded in an BJD-200 ebeam evaporator (Temescal) chamber for gold deposition. Following this step, the wafers were then left overnight in acetone solution to reveal the gold patterns on the biochip.


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To make chambers, a solution of SYLGARD 184 PDMS (polydimethylsiloxane) prepolymer (Dow Corning) was first mixed with its crosslinker in a 10 : 1 ratio, and then poured onto a clean silicon surface for curing at 65 ˚C for 1 h. The cured polymer layer was then punched to create chambers as defined in the biochip design, then removed from the silicon surface and plasma-bonded to the prepared biochip.

Biochip Functionalization Thiolated target-specific forward primers (5 µM, 10 µL) were dropped onto working electrode surfaces within respective amplification/detection chambers, sealed, and incubated overnight in the dark at ambient temperature to form self-assembled monolayers. After electrode surface functionalization, excess unbound primers were removed by washing with phosphate buffer saline (PBS) (10 mM, pH 7.4) solution thrice. Subsequently, 6-mercapto-1-hexanol (1 mM) in glycerol (2.5%)-containing PBS (50 mM, pH 7.4) solution was added to each primer-modifed detection electrode for 30 min at ambient temperature to block bare surfaces prior to PBS washing as described above.

Electrical Release of Cellular Content Cells of cultured cell line and patient sample origins were used for cellular electrical lysis experiments in this study. For PC cell lines, cells were cultured in cultured in RPMI-1640 growth media (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) in a humidified incubator containing 5% CO2 at 37 °C. For patient blood and urine samples, ethics approval was obtained from The University of Queensland Institutional Human Research Ethics Committee (Approval No. 2004000047), and Royal Brisbane & Women’s Hospital Human Research Ethics Committee (Ref No. 1995/088B). Informed consent was obtained from all subjects prior to sample collection and methods pertaining to clinical samples were carried out in accordance with approved guidelines. ACS Paragon Plus Environment

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For electrical cell lysis, a sample (100 µL) containing target cells was loaded into the lysis chamber and exposed to a constant dc potential (4V). Patient whole urine samples were used neat, whilst serum samples were diluted 10-fold with PBS buffer (10 mM) for on-chip lysis. This lysis step was followed by a quick on-chip magnetic wash protocol13 to enrich for total cellular NA in a concentrated volume of 25 µL and remove any inhibitory agents during subsequent target amplification.

Nanofluidic-Enhanced Solid-Phase Isothermal Amplification Aliquots of enriched NA from cell lysate were transferred via built-in fluidic channels into individual amplification/detection chambers for analysis of different genes. For isothermal recombinase polymerase amplification (RPA) of RNA targets, the TwistAmp Basic RT-RPA kit (Twist-DX) was used with slight modifications to manufacturer’s instructions. Briefly, lyzed cellular NA (1.5 µL), each reverse primer (500 nM) (Table S1) and biotinylated dUTPs (20 nM) (Thermo Fisher Scientific) were added to make a reaction volume (6.25 µL) within each chamber prior to incubation at 43°C by placing the biochip on a heating block. Then, nanofluidic enhancement of solid-phase RPA was induced in each chamber through application of an alternating current (ac) field strength of f = 500 Hz and Vpp = 500 mV for 10 min. The ac field parameters were optimized in this study for maximal enhancement effect. Each electrode was then washed with PBS, incubated with streptavidin-paramagnetic iron oxide particles (1 µL) in PBS (10mM, 0.5% triton-X) for 5 min to label biotinylated uracil bases which were incorporated into immobilized amplicons during solid-phase amplification, and washed again with PBS before electrochemical measurements.

Chronoamperometric Electrochemical Analysis For electrochemical detection of immobilized amplicons by chronoamperometry, 1-Step™ TMB substrate solution (15 µL) (Thermo Fisher Scientific) was added to each chamber. At 5 ACS Paragon Plus Environment

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min after TMB-H2O2 addition, H2SO4 (500 mM) was added to stop the reaction and activate TMB for electrochemical detection. Chronoamperometry measurements were carried out using a CH1040C potentiostat (CH Instruments) at 150 mV, 30 s. All measurements were performed at room temperature.

Data Analysis Normalized chronoamperometric signals (X) for each target gene were calculated with notemplate background signal taken into consideration. For example:

XTMPRSS2-ERG = (raw TMPRSS2-ERG signal – no-template control background signal) / notemplate control background signal

(1)

In order to account for different starting amount of prostate cells, TMPRSS2-ERG, PCA3, or SChLAP1 level was calculated by normalizing each target’s chronoamperometric signal to corresponding KLK2 signal for each sample. For example:

TMPRSS2-ERG Level = XTMPRSS2-ERG / XKLK2

(2)

where XTMPRSS2-ERG and XKLK2 are the chronoamperometric signals for TMPRSS2-ERG and KLK2 respectively.

qPCR Validation The KAPA SYBR® FAST One-Step qRT-PCR kit (KAPA Biosystems) was used to set up a single reaction volume for each sample (10 µL). Each reaction volume consist of 1X KAPA SYBR® FAST qPCR Master Mix, each forward and reverse primer (200 nM) (Table S1), 1X KAPA RT Mix, ROX dye (50 nM) and cell line total RNA (30 ng). RT-qPCR was performed ACS Paragon Plus Environment

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using the Applied Biosystems® 7500 Real-Time PCR System (Thermo Fisher Scientific). The cycling protocol was: 42°C for 10 min to synthesize cDNA, followed by 95°C for 5 min before cycling 35 times (95°C for 30 s, 50°C for 30 s and 72°C for 1 min) and finished with 72°C for 10 min.

RESULTS AND DISCUSSION Biochip for Liquid Biopsy Targeted Gene Analysis Our biochip design (Figure S1, Supporting Information) integrated simultaneous ctNA analysis (i.e. sample preparation, target amplification, and detection readout) of four genetic aberrations from liquid biopsies within 30 min. In the central lysis chamber, we begin with on-chip electrical release of cellular material within 60 s by applying an optimized 4V direct current (dc) potential (data not shown) to induce cell membrane pore formation and subsequent cell bursting from osmotic pressure imbalance (Figure 1a; Supplementary Video 1, Supporting Information).18 Next, equal volumes of magnetically-enirched NAs from the cell lysate were transferred into each of the amplification/detection chambers for specfic targeted genetic analysis. Following modern evolution in isothermal amplification techniques,19 we employed solidphase RPA method to streamline target amplification without thermocycling. Using PC as a model, we successfully pre-immobilized each amplification/detection electrode surface with unique forward primer sequences (Figure S2, Supporting Information) for the detection of a PC-specific gene, including: TMPRSS2-ERG gene fusion, PCA3, SChLAP1, or KLK2. Mutations in these genes have been strongly implicated in PC tumorigenesis, and several landmark research studies have showcased their combined clinical analysis value in liquid biopsies for improved PC diagnosis and prognosis.15-17 Each target is captured and amplified in a single step with resultant amplicons being tethered on the electrode surface (Figure 1b). The prime innovative aspect of our biochip lies in the first-time use of customized electrode ACS Paragon Plus Environment

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patterning to create physical nanofluidic enhancement of solid-phase RPA. Under an ac field, the asymmetric circular and ring electrode pattern engendered a nanoscaled fluidic mixing phenomenon13 to promote molecular collision frequency during amplification (Figure 1b). After washing away excess amplification reagents, we attached streptavidin-modified nanozymes to biotin-uracil bases which are pre-incorporated into the surface-bound amplicons during solid-phase RPA. The iron oxide particles function as non-biological peroxidasemimicking nanozymes14 for TMB oxidation (in H2O2 presence) to eventually give two-electron diimine oxidation products. The use of nanozymes confers ideal benefits towards our integrated biochip for rapid targeted gene analysis in a healthcare setting, such as stability from denaturation and inexpensive large-scale synthesis. Chronoamperometry is used to detect the electrochemically-active TMB products,20 and the measured current is correlated to the target gene level (Figure 1c).

Adjustable Nanofluidic Manipulation of Solid-phase Amplification A unique facet of our nanofluidic manipulation is that its fluidic velocity is adjustable over a range of different frequencies (Supplementary Video 2-5, Supporting Information). We found that the most optimal condition for maximal solid-phase RPA enhancement is at 500 Hz and 500 mV amplitude (Figure 2a). Under this condition, we deduced that the nanoscaled fluidic forces were able to manoeuvre biomolecules of similar size range towards the surface-bound primers for faster target hybridization and amplification via increased molecular collisions and reaction kinetics. Crucially, we accomplished solid-phase RPA within an unprecedented 10 min, at which signal saturation was achieved. We also performed a control experiment to test solidphase amplification efficiency in absence of nanofluidic manipulation. We observed that a similar detection signal was only achieved after 50 min when solid-phase RPA is solely dependent on Brownian diffusion (Figure 2b), thus justifying the enhancement effect of nanofluidic manipulation. ACS Paragon Plus Environment

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Specific Targeted Gene Analysis in Cancer Cells Accurate target detection is fundamental for multiple gene target analysis from a high background of interfering biomolecules in liquid biopsies. Our biochip was designed to interrogate four different PC gene targets (TMPRSS2-ERG gene fusion, PCA3, SChLAP1, and KLK2) in separate chambers, and target-specific primer pairs were designed to produce amplicons of different sizes for each target. To evaluate selective target capture and amplification, we subjected each electrode surface-immobilized forward primer set to various well-characterized PC cell lines (DuCap, LnCap, 22Rv1) with known expression of the four target genes. Upon addition of paired reverse primers to initiate target amplification, we found that electrochemically detected target levels in respective PC cell lines corresponded with known expression levels (Figure 3a). Further qPCR profiling and gel electrophoresis of amplicons (Figure S2, Supporting Information) also validated the selective target amplification. Furthermore, nanofluidic forces generated on electrode surfaces would also aid in the removal of weakly-bound non-targets to impart greater target capture selectivity.

Detection Limit

We investigated if the detection limit of our biochip allows for trace NA analysis in liquid biopsies. We titrated in vitro TMPRSS2-ERG targets over a concentration range of 0-1000 copies in a background of TMPRSS2-ERG–negative LnCap NA extract and evaluated the corresponding electrochemical signals (Figure 3b) after solid-phase RPA under nanofluidic manipulation. Our biochip achieved a low 50 copies-detection limit (Figure 3c). We attributed the low detection limit to the combination of isothermal RPA, nanofluidic enhancement, and chronoamperometry readout; all of which have been demonstrated for exceptional detection sensitivity. The close proximity of iron oxide nanozymes and surface-bound DNA amplicons is also beneficial for significantly boosting nanozyme peroxidase-mimicking activity.21 The ACS Paragon Plus Environment

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attained detection sensitivity by isothermal solid-phase RPA is equivalent to that achieved by customary PCR thermocycling. As compared to recent electrochemical chips for cancer celluar NA analysis in clinical samples,13,22,23 our biochip is distinct in empowering an integrated sample-to-answer approach with excellent detection sensitivity due to target amplification, thus making our biochip more amenable for detecting low genetic mutation load. Additionally, our biochip also enabled total integration of sample preparation via on-chip electrical cell lysis and magnetic NA enrrichment as opposed to laboratory-based sample preparation, thus removing the binding of non-specific blood/urine biomolecules which compromise detection performance.

Urinary Targeted Gene Analysis for Cancer Detection and Risk Prediction To demonstrate our biochip for PC diagnosis and risk prediction in patient liquid biopsies, we performed targeted analysis of our four-gene panel in patient samples. As TMPRSS2-ERG gene fusion, PCA3, SChLAP1, and KLK2 have all been previously reported to be present in urine samples,17,24 this form of non-invasive sampling offers an attractive substitute for tissue biopsies without risks of infection and undersampling. In all, we performed simultaneous targeted four-gene analysis in pre-biopsy urine samples from 30 patients who are scheduled for PC biopsies based on unfavourable clinicopathlogical findings and five healthy donors (Figure 4a). To account for varying number of prostate cells in different urine samples, we used KLK2 signals as an internal control to normalize TMPRSS2-ERG, PCA3, SChLAP1 signals, thus ensuring that high detection signals is solely due to abnormal gene overexpression. A target threshold limit was established from the mean signals of healthy donors plus three standard deviations. Altogether, we scored 16 patients (with detected TMPRSS2-ERG, PCA3, SChLAP1 signals over the target threshold limits) with overexpression of the targeted PC genes. Based on biopsy results (Table S2), all 16 patients were detected with prostate tumor growth, and 12 (75%) of the 16 patients were diagnosed with high-grade PC. We also performed quantitative PCR (qPCR) analysis of the four genes from matching patient samples for Passing Bablok ACS Paragon Plus Environment

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regression analysis (Figure S4-6, Supporting Information) against an exisitng laboratory-based technique, and the results indicated that our biochip is a viable alternative approach to qPCR. Our results highlighted the potential of using targeted analysis of multiple PC genetic aberrations in liquid biopsies for accurate PC diagnosis and risk prediction. The rapid on-chip cellular lysis from minuscule volume (100 µL) of urine samples is also beneficial for reducing precious sample requirement; and avoiding lengthy, cumbersome, and laboratory-confined sample preparation steps before genetic analysis.

Matched Patient Urine and Serum Expression Correlation Besides PC screening, we sought to study PC gene expression correlation between serum and urine liquid biopsies of a single patient. Both blood and urine liquid biopsies are emerging as compelling sources for non-invasive PC detection4 but there are currently no systematic comparisons between both these forms of liquid biopsies. Thus, a correlation study of matched urine and serum samples may aid in evaluating the clinical feasibility of using either/both liquid biopsy sources for targeted gene analysis. In a cohort of 20 PC patients, we performed TMPRSS2-ERG, PCA3, SChLAP1, and KLK2 analyses in serum and urine (Figure 4b). Both liquid biopsy forms yielded sufficient cellular material for analysis after electrical release on our biochip, and the four-target expression profiles were overall congruent in both serum and urine samples (Pearson correlation coefficient = 0.87). Notwithstanding the limited cohort size, this result supported the possibility of using our biochip system for use with patient serum and/or urine PC liquid biopsies.

Cancer Relapse Monitoring As a final demonstration of our biochip’s multifunctional capabilities, we also investigated our biochip’s clinical potential as a PC relapse monitoring tool in liquid biopsies. Non-invasive relapse monitoring is feasible for patients able to sequentially provide urine/blood samples at ACS Paragon Plus Environment

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minimal health risk and cost.3,25 To this end, our biochip offers the benefit of avoiding serial invasive biopsies during longitudinal relapse monitoring, and in patients with non-accessible metastases such as bone-predominant lesions of advanced PC. Using pre- and post-treatment urine samples, we performed targeted gene analysis in five PC patients who underwent radical prostatectomy treatment (Figure 4c). Two of the five patients were identified with no significant decrease in overexpressed TMPRSS2-ERG, PCA3, SChLAP1 levels after undergoing treatment, and these outcomes were correlated with PC biochemical relapse. In contrary, the remaining patients displayed low gene expression of the four targets and remained relapse-free. These results highlighted the potential usefulness of our biochip for treatment monitoring by providing rapid outcomes from repeated liquid biopsies.

CONCLUSIONS In sum, we have developed an integrated biochip with for targeted gene analysis in patient liquid biopsies. Crucially, the biochip permitted an entire 30 min sample-to-answer workflow empowered by on-chip electrical cell lysis and nanofluidic enhancement of solid-phase target amplification through increased molecular collisions. This rendered our approach more practical for clinical liquid biopsy applications by reducing analysis time, cost and starting sample amount as compared to current techniques. In overcoming the challenges of ctNA detection in liquid biopsies, we anticipate that our biochip may essentially be used for broad NA detection applications in different diseases.

ASSOCIATED CONTENT Supporting Information Available: The following files are available free of charge. This material includes detailed biochip design, primer sequences, electrochemical characterization of functionalized electrode, gel electrophoresis of amplicons, pathological data on patient biopsy samples, and Passing-Bablok analyses.


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Conflict of Interest Disclosure The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors acknowledge grants received by our laboratory from the National Breast Cancer Foundation of Australia (CG-12-07), the ARC DP (140104006) and the ARC DP (160102836). These grants have significantly contributed to the environment to stimulate the research described here. This work was performed in part at the Queensland node of the Australian National Fabrication Facility. A company established under the National Collaborative Research Infrastructure Strategy to provide nano and microfabrication facilities for Australia’s researchers. K.M.K acknowledges support from the Australian Government Research Training Program Scholarship. We thank Robert ‘Frank’ Gardiner and Aine Farrell of the University of Queensland Centre for Clinical Research for providing patient urine and serum samples.


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Figure 1. Integrated biochip (with a central lysis and four individual amplification/detection chambers linked by fluidic channels) for targeted gene analysis in liquid biopsies. a) On-chip electrical cell lysis to release cellular targets within 60 s. b) Nanofluidic manipulation under an ac field to enhance isothermal solid-phase amplification of targets. c) Electrochemical detection of surface-immobilized amplicons via superparamagnetic iron oxide particle nanozymemediated redox reaction.


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Figure 2. Adjustable nanofluidic manipulation of solid-phase amplification. a) Optimization of frequency (f) (0-1000 Hz) at constant amplitude (Vpp) of 500 mV to determine the optimal applied field strength. b) Improvement in amplification time to achieve detectable signal under nanofluidic enhancement. Error bars represent standard deviations of three technical replicates.


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Figure 3. Analytical performance of biochip. a) Detection selectivity in profiling gene expression in PC cell lines. b) Corresponding chronoamperometric signals of 0-1000 copies of TMPRSS2-ERG targets in a background of TMPRSS2-ERG–negative LnCap NA extract. c) Detection limit of 50 target copies. Error bars represent standard deviations of three technical replicates.


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Figure 4. Multifunctional clinical capabilities of integrated biochip. a) Targeted expression level analysis of four genes in urine samples for PC diagnosis and risk prediction (n = 35). Heat map represents KLK2-normalized expression levels for TMPRSS2-ERG, PCA3, and SChLAP1. “P” denotes “PC Patient” and “H” denotes “Healthy Donor”. “*” represents overexpression in targeted genes. b) Correlation study of biomarker expression in matching patient urine and serum samples (n = 20). c) Non-invasive pre- and post-treatment targeted gene profiling in patient urine samples for PC relapse monitoring (n = 5).


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A Sample-to-Targeted Gene Analysis Biochip for ... - ACS Publications

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