Microfluidic Validation of Diagnostic Protein Markers for Spontaneous

Article pubs.acs.org/jpr

Microfluidic Validation of Diagnostic Protein Markers for Spontaneous Cerebrospinal Fluid Rhinorrhea Akwasi A. Apori,† Martina N. Brozynski,† Ivan H. El-Sayed,‡ and Amy E. Herr†,* †

Department of Bioengineering, University of CaliforniaBerkeley, Berkeley, California Department of Otolaryngology, University of CaliforniaSan Francisco, San Francisco, California

‡

S Supporting Information *

ABSTRACT: Cerebrospinal fluid (CSF) rhinorrhea is a potentially dangerous condition identified by CSF leakage into the nasal cavity. This malady stands to benefit from rapid and noninvasive screening diagnostics to complement low-throughput imaging based methods currently in use. To address this gap, we demonstrate on-chip immunosubtraction to accelerate biomarker validation and immunoassay development for a putative CSF rhinorrhea diagnostic marker, transthyretin, by combining high-specificity immunoaffinity capture with subsequent polyacrylamide gel electrophoresis (PAGE). We demonstrate the on-chip assay using photopatterned polyacrylamide immunofilters. The filter consists of polymer with controlled pore-sizes to size-exclude (i.e., “subtract”) large antibody-target immune complexes from downstream PAGE separation. A control PAGE separation is also performed for comparison without immunoaffinity capture (i.e., no antibody present). We compare on-chip immunosubtraction to Western blotting and ELISA to validate CSF rhinorrhea biomarkers from nasal surgery samples. For samples representative of spontaneous rhinorrhea, the 5 min on-chip assay achieved clinical specificity of 100%, compared to 50% for ELISA which required 6 h. On-chip immunosubtraction also generated results for clinical samples not assayable via ELISA due to matrix protein spurious signals. The pilot study suggests the capability of a rapid on-chip validation tool to expedite scrutiny of putative protein markers for new clinical assays. KEYWORDS: cerebrospinal fluid rhinorrhea, immunosubtraction, transthyretin, beta-2-transferrin, biomarker validation, traumatic brain injury, microfluidic, electrophoresis

■

fixation electrophoresis and ELISA can also confirm the presence of CSF in many cases;5,6 therefore, most diagnostic algorithms recommend initially performing noninvasive biomarker screening assays.5 A positive screening result would thus justify more thorough otalaryngologic examination to locate and visualize possible defects for surgical treatment. 5 Unfortunately, the lack of available rapid biomarker screening methods can lead practitioners to opt directly for unnecessary surgical exploration and reparative procedures for rhinorrhea pathologies falsely presumed to be caused by CSF leakage.5,7 One frequently used confirmatory technique couples high resolution CT scans with endoscopy including intrathecal application of fluorescein dye for leakage localization. This diagnostic approach can have serious side effects since endoscopic nasal procedures create potential for further tissue damage.8 Coupled with this risk, addition of fluorescein to the intracranial cavity is not manufacturer nor FDA approved and at sufficient doses has been correlated to grand mal seizures and spinal headaches.8 Due to the morbidity risk of invasive techniques, CSF

INTRODUCTION CSF rhinorrhea occurs when CSF from the cranial cavity leaks into the nasal cavity. Etiologies are diverse and include head trauma, neurosurgery, invasive tumors, congenital defects, and nontraumatic spontaneous leakage or elevation of intracranial pressure (repetitive strenuous exercise).1−3 Symptoms classically include drainage of a low-viscosity, clear, watery unilateral or bilateral nasal secretion that increases outflow as the patient leans forward.1 CSF rhinorrhea is a result of disruption of the brainsinus barrier that can lead to serious and potentially deadly complications including bacterial meningitis (10% risk annually, 40% long-term) or viral infection, intracranial hypotension, pneumocephalus, anosmia, frontal lobe abscess or even stroke.2−4 Therefore, early detection and treatment of CSF rhinorrhea are critical to patient prognosis and outcome. Diagnosis currently relies on a combination of detection methods including computed tomography (CT) scan, magnetic resonance imaging (MRI), and endoscopic examination. Often, diagnosis includes invasive techniques requiring puncture of the lumbar spine and injection of radioactive or contrast dye used with radionuclide cisternography or CT cisternogram to image leakage of the fluid. Laboratory analyses including immuno© 2013 American Chemical Society

Received: October 2, 2012 Published: January 23, 2013 1254

dx.doi.org/10.1021/pr300928p | J. Proteome Res. 2013, 12, 1254−1265

Journal of Proteome Research

Article

accessible and amenable to routine screening diagnostics than those used for previous studies collected via neurosurgery or lumbar puncture.19,30,31 Herein we detail use of an automated microfluidic tool for proteomic analysis of raw nasal secretions from a clinical cohort of patients undergoing otolaryngologic treatment at the University of California San Francisco Medical Center. Assays are performed in 5−10 min using the on-chip immunosubtraction technique as opposed to 4−10 h for comparable diffusion based techniques32 (i.e., Sepharose beads with PAGE). Among the collected samples, we identify those representative of spontaneous CSF rhinorrhea to validate putative CSF biomarkers (TTR and β2TF) differentiating head trauma from standard proteomic content in raw nasal discharge. Analysis is performed using the on-chip immunosubtraction instrument to perform qualitative assays for detection of CSF rhinorrhea as well as quantitative assessment of the TTR biomarker concentration. Comparison to benchtop methods for qualitative assessment (Western blot) and biomarker quantitation (TTR ELISA) were also performed to benchmark the on-chip biomarker validation technique compared to traditional methods.

rhinorrhea diagnoses would benefit from an automated, rapid, and noninvasive protein biomarker based nasal discharge screening assay for CSF-specific proteins that can be implemented prior to more invasive tests (e.g., CT cisternogram) to localize leaks for repair.5,9 Recent proteomic studies highlight the diagnostic potential of two putative biomarkers, beta2-(tau)-transferrin and transthyretin (β2TF and TTR), in detecting CSF rhinorrhea. While these proteins hold promise for differentiating CSF from nasal mucus using laboratory electrophoresis assays (i.e., twodimensional electrophoresis, Western blotting),4,10 most reports on assessing aqueous rhinorrhea to date have focused exclusively on detecting β2TF or β-trace protein.6 However reported false positives and false negatives as well as slow assay times (>1 h) point to the utility of introducing additional biomarkers such as TTR into a more reliable and rapidly assessable biomarker panel diagnostic.4,11,12 Additionally, commercial β2TF assays are only performed by a few laboratories in the U.S. requiring doctors to send out samples for analysis and requiring many days to receive results. As a diagnostic biomarker, TTR is more amenable to electrophoretic detection than β2TF because TTR has a significantly faster mobility than larger interfering matrix proteins (i.e., albumin and lactoferrin). However, to our knowledge TTR has yet to be included as an individual or panel biomarker in any commercial CSF rhinorrhea diagnostics. Immunosubtraction is a powerful electrophoretic laboratory medicine assay reporting both protein mobility and binding specificity of target analytes which is well suited for biomarker detection within CSF, mucus, or other complex biological matrices.13,14 Presently, lab medicine immunosubtraction is typically implemented via antibody-based bead capture used to ‘subtract’ a target analyte from subsequent native polyacrylamide gel electrophoresis (PAGE).15−18 Identification of target analytes is then performed by comparing PAGE electropherograms run with and without target subtraction.13,14 A major shortcoming of conventional slab gel immunosubtraction for diagnostic biomarker validation is the long assay duration (2−4 h) and concomitant low throughput. Recently, we have reported integration of immunosubtraction into a single instrument platform amenable to high throughput CSF analysis using microfluidic technology;18,19 while our group and others have also addressed the need for increased automation and speed for integrated proteomic tools.20−23 The microfluidic approach automates several required sample preparation steps including sample incubation and mixing, analyte enrichment, antibody− antigen binding, and fluorescence labeling to detect putative biomarkers via immunosubtraction electrophoresis.19 Advanced in situ photopolymerization techniques are essential to integrating sample preparation with analytical functions harnessing small transport length scales within integrated architectures.19,24,25 Precise functionalized gel regions are fabricated contiguously within the glass microchannel geometry with UV photolithography to form discrete reaction zones with unique physical properties (e.g., polymer cross-linking ratio to control pore size). Several reports from our group and others have approached various aspects of integrated sample preparation and analysis with functionalized PA gels;26−29 however, to our knowledge, no previous reports have shown a microfluidic assay to diagnose CSF rhinorrhea. This report is the first using the new homogeneous on-chip immunosubtraction tool for analysis of putative CSF rhinorrhea biomarkers from raw nasal secretionssamples more easily

■

MATERIALS AND METHODS

Reagents and Proteins

Glacial acetic acid, 3-(trimethoxysilyl)propyl methacrylate (98%), and 30%T (29:1) acrylamide/bis-acrylamide solutions were purchased from Sigma-Aldrich (St. Louis, MO). A water-soluble photoinitiator 2,2′-azobis[2-methyl-N-(2hydroxyethyl)propionamide] (VA-086) was purchased from Wako Chemicals (Richmond, VA). 10× Tris/glycine native electrophoresis buffer (25 mM Tris, pH 8.3, 192 mM glycine) was purchased from Bio-Rad (Hercules, CA). Ladder proteins including TTR and human serum albumin (HSA) along with polyclonal antibodies to TTR and HSA were purchased from Life Technologies (Carlsbad, CA). Monoclonal antibodies for beta-2-transferrin, Prostaglandin-D synthase, and secondary HRP conjugated antirabbit and antimouse antibodies were purchased from Abcam (Cambridge, MA). With the exception of on-chip labeling experiments, unlabeled proteins were fluorescently labeled in-house using Alexa Fluor 488 protein labeling kits (Life Technologies, Carlsbad, CA). Purification of labeled proteins was performed with P6̅ and P-30 Bio-Gel columns (Bio-Rad, Hercules, CA). Postlabeling, proteins were diluted with 1× Tris/glycine native buffer to attain desired concentrations. Nasal discharge samples run on-chip were diluted 10× using 1× Tris/glycine to prevent detector saturation while CSF samples were undiluted. On-chip labeling was conducted using reagents from the CE 503 dye kit from Active Motif (Eugene, OR). The CE 503 dye reagent was mixed with 1× Tris/glycine buffer at a ratio of 1:4. Proteins were stored at 4 °C in the dark until use. For off-chip sample preparation, protein-antibody complexes were formed by incubating the target protein with the relevant antibody for at least 1 h at room temperature. SDS-PAGE and Western blot assays of biological samples were performed using Novex 4−12%T and 4−20% Tris/glycine precast gels with the XCell Surelock Mini-cell gel electrophoresis system (Invitrogen, Carlsbad CA) for separation and blotting to PVDF membranes. Western Lightning enhanced chemiluminescent substrate (HRP catalyzed) was purchased from Perkin-Elmer (Waltham, MA). TTR direct sandwich ELISA kits were purchased from TSZ ELISA (Framingham MA) and CUSAbio 1255



Article

UV illumination sourcea 4× UV objective on a Nikon microscope in conjunction with an adjustable power mercury arc lamp (Hamamatsu, Bridgewater NJ).18 The 3%T PA precursor solution was then used to replace the unpolymerized 12%T precursor solution through a vacuum purge to form the loading gel. The entire unmasked chip was then exposed to UV flood illumination for 10 min at approximately 18 cm from a 100W UV lamp (UVP B100-AP, Upland, CA) at ∼10 mW/cm2.

(Wuhan, P. R. China). Tau (β2TF) indirect sandwich ELISA kits were purchased from Invitrogen and CUSAbio. Clinical Samples

Blinded clinical specimens were collected pre- and postoperatively at the UCSF Medical Center from patients undergoing sinus or neurological surgery under the approval of the UCSF institutional review board. All patients consented to participation in the study prior to sample collection and clinical samples were deidentified prior to analysis in the Herr Lab at UC Berkeley. Some mucus samples were mixed with nasal lavage fluid during collection. All samples were stored at 2−8 °C for up to 24 h immediately after collection prior to storage at −25 °C until assayed. Pooled human CSF baseline samples obtained via lumbar puncture from healthy donors were purchased from Biological Specialty Corporation (Colmar, PA). Pooled human nasal mucus and saliva standards were collected directly from healthy volunteers not suffering from rhinorrhea.

Apparatus and Imaging

Disposable pipet tips (200 μL) were cut at the ends and inserted into the chip wells to form loading reservoirs. To conserve sample, 5 μL of sample was loaded into the sample well while 60 μL of 1× Tris/glycine native buffer was loaded into all other wells. High voltage for the electrophoretic transport of reagents was applied with a custom-built, programmable high-voltage power supply (0 to 3000 V output range) using platinum wire electrodes. An inverted epifluorescence microscope was used for imaging (IX-71, Olympus, Melville, NY) in conjunction with a 10× objective (NA 0.3) and filter cubes optimized for FITC and Texas Red detection. A 1004 × 1002 Peltier-cooled EMCCD camera (iXon+ 885, Andor Technologies, South Windsor CT) was used to record images of migrating proteins. 250 ms exposure images were captured with 4× pixel binning applied in the direction transverse to separation. Image J software was used for postcapture analysis (National Institutes of Health, http:// rsbweb.nih.gov/ij/). Background images were subtracted to account for variability of the light source. Average fluorescence intensity in a region of interest (ROI) occupying the channel width at a fixed axial position (ROI ≈ 160 × 80 μm) approximately 1.5 mm downstream from the immunosubtraction filter interface was used to plot electropherograms. Run-to-run variability was normalized to either total fluorescence or a free-dye internal standard.

Chip Fabrication & Surface Preparation

Soda-lime glass microfluidic chips were purchased from Caliper Life Sciences (Hopkinton, MA). The chip design consisted of four wells used for immunosubtraction (sampleS, sample wasteSW, bufferB, and buffer wasteBW) plus three additional reagent/buffer wells all connected to a separation channel that was 2.05 mm in length, ∼80 μm wide, and 15 μm deep (Figure 1A). The glass channel walls were cleaned prior to in situ gel polymerization by washing with 1 M NaOH for 5 min to remove debris followed by a deionized water wash to remove NaOH. Channel walls were functionalized to enable covalent linkage to PA by using capillary action to load a 2:3:5 ratio solution of 3-(trimethoxysilyl)propyl methacrylate, glacial acetic acid, and deionized water.25,28 The surface preparation solution was allowed to incubate for 30 min prior to vacuum purging and flushing channels twice each with 30% glacial acetic acid followed by deionized water.

ELISA and Western Blot Protocol

On-Chip Immunofilter Fabrication

Standard protocols were followed for all ELISA kits (TTR direct competitive sandwich ELISA, β2TFindirect sandwich ELISA). All samples were diluted to appropriate concentrations within the standard range using diH20. The 96-well plate was read using O.D. 450 nm measurements in an automated plate reader (Tecan Infinite m200 Pro, Tecan Group Ltd., Switzerland). Western blot was performed by first running SDS-PAGE in the Invitrogen Xcell Surelock Mini-cell with 4− 20%T precast Tris/glycine gels. 125 V was applied for 90 min for separation in a 1× Tris/glycine/SDS running buffer. Blotting onto 0.2 μm pore-size PVDF membranes (Invitrogen) was done using the Biorad Xcell II Blot Module to apply 25 V for 75 min in 1× transfer buffer. Membranes were blocked with 50 mg/mL BSA overnight prior to following standard Western blot antibody incubation and wash protocols (see Supporting Information S.2 for details). Duplicate membranes were stained with Coomassie Brilliant Blue for protein band reference images. Imaging was performed using the ChemiDoc XRS+ system (Bio-Rad, Hercules, CA).

The molecular weight cutoff immunofilter and PAGE gel were defined at the start of the electrophoretic separation channel by creating a sharp step decrease in polyacrylamide (PA) gel poresize. PA gel precursor solutions used to photopattern immunofilters in situ were made by diluting the total volume of the 30%T 3.3%C (w/v) acrylamide/bisacrylamide solution to the necessary ratio and adding 0.2% (w/v) VA-086 photoinitiator (cautionacrylamide monomer is a potent neurotoxin requiring proper safety precautions). Prior to loading on-chip, PA gel precursor solutions were sonicated and degassed 5−10 min. 12%T PA gel filters (total acrylamide %T, with 3.3% w/w cross-linker, %C) were chosen for experiments and empirically determined to have a pore-size cutoff near 150 kDa, making this pore-size effective for subtraction of TTR-antibody complexes (∼205 kDa). A 3%T loading gel was polymerized contiguous to the 12%T gel creating a step discontinuity from large to small pore-size between the loading gel and filter region. The discontinuity allowed sample stacking as proteins migrated across the interface for improved resolution and unbiased initiation of the protein separation step. Photolithography was performed using a film transparency mask containing a 500 μm × 4 mm opening (Fineline Imaging, Colorado Springs, CO) which was aligned with the desired channel containing 12%T precursor solution then exposed to UV power (∼5.8 mW/cm2) for 4 to 5 min from a collimated

Mass Spectrometry Protocol

CSF, mucus, and clinical samples were prepared for mass spectrometry by removing salts, detergents, and other nonprotein contaminants using a 10 kDa molecular weight cutoff Amicon Ultra centrifugal filter (Millipore, Billerica MA). Proteins were reconstituted with diH20 then precipitated 1256



Article

Figure 1. Rapid on-chip immunosubtraction integrates into the biomarker validation pipeline. (A) For immunosubtraction, a 7-well glass microfluidic chip with an offset t-channel geometry was used to permit fixed volume sample injection for downstream electrophoresis with additional reagent wells for on-chip sample preparation. (B) The on-chip immunosubtraction chip contains a microchannel network patterned with in situ polymerized PA gel regions with various pore sizes to serve as size-exclusion filters to large antibody−antigen complexes. Samples are separated electrokinetically across the immunofilter and imaging region (1.5 mm downstream detection). Detection of target analyte is performed via comparison of control (no Ab) and immunosubtraction (+ Ab) PAGE where the target analyte is excluded at the discrete border between the 3%T loading gel and 12%T immunofilter. (C) On-chip immunosubtraction integrates into the biomarker validation pipeline combining the validation assay development with diagnostic assay development on a single automated, inexpensive, and high-throughput platform. (* indicates current methods for CSF rhinorrhea detection).

mass spectrometry protocol). Results were analyzed using inhouse software provided by the Coates Proteomics Laboratory.

using 20% trichloroacetic acid. Precipitated proteins were resuspended in a Urea solution (8 M urea, 100 mM Tris) prior to trypsin enzymatic digestion. Prepared samples were sent an external facility for analysis (QB3 Vincent J. Coates Proteomics/Mass Spectrometry Laboratory, Berkeley, CA). Analysis was performed via 1-D reverse phase separation on a nanoscale HPLC column with subsequent tandem mass spectrometry (see Supporting Information S.4 for detailed

Assay Operation and Validation Study Experimental Protocol

To operate the immunosubtraction assay, a 2 μA current was applied from the sample well (S) to the sample waste (SW) through a large pore size polyacrylamide gel (3%T, 3.3%C) to 1257



Article

clinical patient CSF sample representative of CSF rhinorrhea. The baseline CSF was a pooled sample collected via lumbar puncture from donors. The baseline mucus was a pooled sample collected from healthy donors. The clinical CSF sample was a representative patient sample collected interoperatively during surgery. The Venn diagram in Figure 2 shows the results

introduce the sample proteins in a controlled manner into the fixed injection volume delineated zone for downstream injection while suppressing electroosmotic flow (∼3 min loading time). After sample loading was complete, the sample was electrophoresed (3 μA current applied) to the immunosubtraction PAGE region where proteins were transported across a discrete step decrease from a large pore-size loading gel (3%T, 3.3%C) to a small pore-size separation gel (12%T 3.3%C) as shown in Figure 1B (∼2 min separation time). This interface acted as a size-based immunofilter excluding large proteins and capture antibodies over 150 kDa. Thus any target analyte binding to the capture antibody formed a large complex excluded from subsequent PAGE analysis. Comparison of control (no Ab) and immunosubtraction (+Ab) electropherogram profiles imaged downstream (∼1.5 mm) via single point fluorescence detection allowed identification and quantitation of biomarker content within complex biological matrices. Onchip immunosubtraction assay protocols have been previously optimized18,19 and are described in further detail in the Supporting Information S.1. The on-chip immunosubtraction assay was designed to provide not only rapid and portable qualitative data collection for protein identification in complex biological matrices, but also high-throughput biomarker quantitation required for validation studies. The immunosubtraction tool was implemented as a rapid TTR validation screening assay on clinical samples to expedite the CSF rhinorrhea biomarker validation pipeline (Figure 1C). Incorporating microfluidic immunosubtraction into the biomarker validation protocol obviates the need for independent development of separate validation and diagnostic assays. The validation protocol algorithm commenced with analyzing representative samples of the biological matrices under study, including CSF and mucus, via mass spectrometry to confirm presence of putative biomarkers and verify efficacy of the clinical sample collection technique. The on-chip method was then compared to qualitative (Western blot) and quantitative (ELISA) benchtop biomarker validation methodsused in early biomarker validation stagesto quantify protein markers from the clinical samples suitable for a high-throughput single or multiple biomarker diagnostic assay. For calculating assay sensitivity and specificity, only nonturbid spontaneous rhinorrhea representative samples (i.e., minimal blood contamination) including clinical CSF, baseline CSF, and mucus were consideredcomprising a total pool of 11 samples. Slab gel Western blotting results (TTR detection) for clinical samples were taken as the definitive measurement for CSF presence in samples (i.e., true positive determination). Sensitivity and specificity for the on-chip or ELISA techniques were calculated as follows:

■

sensitivity =

#truepositives #truepositives + #falsenegatives

specificity =

#truenegatives #truenegatives + #falsepositives

Figure 2. Venn diagram showing proteins identified via LC/MS in a pooled baseline CSF sample, a pooled baseline mucus sample, and a clinical patient CSF sample. From the CSF baseline sample 103 total proteins with 12 unique proteins were detected. Within the nasal mucus baseline sample 93 total proteins with 61 unique proteins were identified. The clinical CSF sample contained 207 total proteins of which 111 were unique.

of total protein identification among the three samples. In the baseline CSF sample, 103 proteins were detected of which 12 were unique. Ninety-three proteins, of which 61 were unique, were detected in the baseline mucus sample and 207 total proteins with 111 unique were identified in the CSF clinical sample. The relative abundance of the proteins identified in each sample matrix was also determined via mass spectrometry by dividing the spectrum count by the sequence length for each protein. Table 1 shows the 15 most abundant proteins in each sample matrix. For the baseline CSF sample albumin was most abundant composing almost 38% of the total protein while putative CSF rhinorrhea diagnostic biomarkers β2TF, prostaglandin-D-synthase (PGDS), and TTR were the next most abundant proteins all with concentrations near 9% of total protein. The most abundant protein in the baseline mucus sample was lactoferrin (20%) while lysozyme C and putative mucus biomarker PIP were the next most abundant proteins at 18% and 14% respectively. Within the clinical CSF sample serum albumin was most prevalent at 17% followed by 3 haptoglobin isoforms ranging from 6 to 3% while β2TF was the fifth most abundant protein at 3% of the total. TTR was also present at 2% in the clinical sample as the 12th most prevalent protein. There were differences in both the total number of unique proteins identified in the clinical CSF sample which increased over the baseline CSF sample and the relative prevalence of target biomarkers (TTR, β2TF, and PGDS) which decreased in the clinical sample due to the collection method and associated contamination from proximal fluids (i.e., mucus, saliva, blood). A key result pointing to the

RESULTS

Matrix Protein Identification via Mass Spectrometry

In order to validate the presence of TTR and other potential biomarkers of interest in samples used in this study, reversedphase HPLC with mass spectroscopy was run on representative baseline pooled samples for CSF and mucus in addition to a 1258



Article

Table 1. LC/MS Matrix Protein Identification for CSF, Mucus, and Clinical CSF Samples sample CSF baseline

rank

descriptive name

MW (Da)

SC/L

% total

1

serum albumin preproprotein serotransferrin precursor prostaglandin-H2 Disomerase transthyretin precursor alpha-1-antitrypsin precursor immunoglobulin lambdalike polypeptide 5 alpha-1-acid glycoprotein 1 precursor apolipoprotein D precursor haptoglobin isoform 2 preproprotein apolipoprotein A-I preproprotein apolipoprotein E precursor haptoglobin isoform 1 preproprotein cystatin-C precursor beta-2-microglobulin precursor alpha-1-acid glycoprotein 2 precursor lactotransferrin precursor lysozyme C precursor prolactin-inducible protein precursor lipocalin-1 precursor long palate, lung and nasal epithelium carcinomaassociated protein 1 precursor polymeric immunoglobulin receptor precursor zinc-alpha-2-glycoprotein precursor cystatin-S precursor

69367

4.348112

37.946

77050 21029

1.075931 1.073684

9.390 9.370

15887 46737

1.013605 0.440191

8.846 3.842

23063

0.280374

2.447

23540

0.243781

2.127

21276

0.164021

1.431

38452

0.118156

1.031

30778

0.116105

1.013

36154 45205

0.107256 0.105911

0.936 0.924

15799 13715

0.10274 0.10084

0.897 0.880

23603

0.099502

0.868

78182 16537 16572

3.214085 2.959459 2.239726

19.820 18.250 13.812

19250 52442

1.107955 0.917355

6.832 5.657

83284

0.486911

3.002

34259

0.412752

2.545

16214

0.390071

2.405

2 3 4 5 6 7 8 9 10 11 12 13 14 15 mucus baseline

1 2 3 4 5

6 7 8

sample

clinical CSF

rank

descriptive name

9 immunoglobulin J chain 10 zymogen granule protein 16 homologue B precursor 11 immunoglobulin lambdalike polypeptide 5 12 serum albumin preproprotein 13 cystatin-SN precursor 14 beta-2-microglobulin precursor 15 clusterin isoform 2 preproprotein 1 serum albumin preproprotein 2 haptoglobin isoform 2 preproprotein 3 haptoglobin isoform 1 preproprotein 4 haptoglobin-related protein precursor 5 serotransferrin precursor 6 alpha-1-antitrypsin precursor 7 immunoglobulin lambdalike polypeptide 5 8 serum amyloid A protein preproprotein 9 prostaglandin-H2 Disomerase 10 cystatin-C precursor 11 hemoglobin subunit beta 12 ribonuclease pancreatic precursor 13 transthyretin precursor 14 serum amyloid A2 isoform b 15 serum amyloid A2 isoform a

MW (Da)

SC/L

% total

18099 22739

0.352201 0.350962

2.172 2.164

23063

0.327103

2.017

69367

0.321839

1.985

16388 13715

0.297872 0.193277

1.837 1.192

52495

0.091314

0.563

69367

2.73399

16.701

38452

0.9683

5.915

45205

0.916256

5.597

39030

0.548851

3.353

77050 46737

0.498567 0.461722

3.046 2.820

23063

0.457944

2.797

13532

0.409836

2.504

21029

0.384211

2.347

15799 15998 17644

0.342466 0.333333 0.032051

2.092 2.036 0.196

15887 9184

0.306122 0.289157

1.870 1.766

13527

0.229508

1.402

buffer with Ab) was achieved by addition of the same anti-TTR Ab (0.17 mg/mL). The native mobilities of major matrix proteins observed during immunosubtraction electrophoresis were also characterized to validate protein identity (Figure 3B). Current controlled separation (3 μA, ∼600 V/cm) of undiluted baseline CSF and baseline mucus (10× dilution) was performed with single point detection 12 mm downstream in the immunofilter. Two major peaks were observed in the mucus samples, a narrow bandwidth PIP peak with apparent electrophoretic mobility of ∼8.00 × 10−9 m2/(V s) and a broad LF peak with apparent mobility of ∼2.94 × 10−9 m2/(V s). The CSF sample provided a distinct peak migration profile distinguishing the CSF sample from the mucus sample. Within the CSF sample the major peaks identified included a narrow bandwidth TTR peak with apparent mobility of ∼5.26 × 10−9 m2/(V s) and a broad comigrating HSA and β2TF peak with apparent mobility of ∼3.51 × 10−9 m2/(V s). The peak area signal-to-noise (S/N) ratios for the largest peaks in baseline mucus and CSF were, respectively, S/N = 2381 and 11667; these high signals resulted from the stacking effect of proteins as they migrated from the large pore-size loading gel to the small pore-size immunofilter. In both the model protein system and endogenous CSF or mucus samples, detection was achieved in less than 2 min, demonstrating the ability for rapid and effective qualitative

efficacy of TTR as a CSF rhinorrhea biomarker was that TTR was identified uniquely in the baseline and clinical CSF samples whereas transferrin was present in all three samples, although at low concentration (0.02%) in the baseline mucus (see Supporting Information Table S-7). On-Chip CSF Rhinorrhea Biomarker Quantitation

After validating the presence of TTR in CSF samples via mass spectrometry, the on-chip immunosubtraction assay provided detection of target biomarkers by two means: (1) antibody capture of target analytes and (2) native mobility assessment of the migrating target during subsequent PAGE. Optimization of both steps was crucial for high-confidence unambiguous biomarker detection.19 To verify TTR binding affinity to the selected polyclonal antibody under assay conditions, a representative two-protein model system was tested for TTR capture capability (Figure 3A). When compared to the control run with no antibody, results indicated that in a 1× Tris/glycine buffer model system 100% of the TTR peak (0.01 mg/mL) was subtracted by addition of polyclonal anti-TTR Ab (0.0375 mg/ mL) while the nonspecific protein S100 was unaffected. Subsequently, this selective polyclonal antibody was used to immunosubtract TTR present in the endogenous baseline CSF sample. Selective subtraction of 95% endogenous TTR from a 90% CSF sample (diluted 1/10 in 1× Tris/glycine 1259



Article

Figure 3. On-chip homogeneous immunosubtraction assay enables qualitative and quantitative detection of putative protein biomarker (TTR) from complex biological fluids. (A) Antibody-target detection was demonstrated via efficient PAGE immunosubtraction of TTR from a model system (S100 and TTR in 1× Tris/glycine) resulting in 100% peak subtraction while subtraction of 95% endogenous TTR from a pooled human CSF sample was achieved in 2 min. (B) On-chip immunosubtraction PAGE of baseline CSF and mucus samples demonstrate qualitative detection of major matrix proteins and putative biomarkers via native electrophoretic mobility assessment of migrating protein bands (separation length is 12 mm). High signal-to-noise was observed for both baseline CSF and mucus samples (*nasal mucus diluted 10 × ) which displayed distinct biomarker profiles observed after 75 s. (C) The calibration curve for TTR quantitation showed that the CSF clinical range is within the linear detection range for the on-chip immunosubtraction assay.

peak S/N = 128 for undiluted CSF and S/N = 187 for undiluted nasal mucus after 5 h separation and Coomassie staining. Several peaks were resolved for the two baseline samples run with slab gel SDS-PAGE (CSF ∼4 peaks, nasal discharge ∼7 peaks). Projected identification of peaks was made based on mobility and relative concentration resulting in purported identity of the four most prevalent proteins in each baseline sample. The most abundant CSF baseline proteins (PGDS, TTR, HSA, and β2TF) and mucus baseline proteins (LYZ, PIP, LNEC, and LF) were distinct between the two samples. Patient clinical samples were also assessed via Western blot to determine presence of TTR. Clear (nonbloody) samples representative of potential spontaneous rhinorrhea were separated by SDS-PAGE and transferred to PVDF membranes for subsequent staining with Coomassie Brilliant Blue (Figure 4C) or Western blot enhanced chemiluminescent substrate detection (Figure 4D). Grossly hemolyzed (bloody) samples were not included in the analysis as they were not representative of spontaneous rhinorrhea manifestation for which the on-chip assay was designed (see Supporting Information Figure S-3). TTR mobility was observed from lane 1 which contained a purified TTR standard (200 μg/mL) which migrated primarily in tetramer form (55 kDa) with some smaller and larger isoforms present. Lanes 2, 5, 7, and 10 contained samples visually characterized as CSF upon collection during surgery while lanes

protein target identification via both binding affinity (i.e., peak subtraction) and native mobility measurements. To demonstrate TTR quantitation capability, a calibration curve for the on-chip immunosubtraction assay was developed. A series dilution of purified TTR protein (in 1× Tris/glycine) was analyzed on-chip in triplicate for peak area quantitation (Figure 3C). A standard regression was fitted to the data using the Hill dose−response model with variable Hill coefficient resulting in a typical sigmoidal assay response (y = A1 + (A2 − A1)/(1 + 10(LOGx0‑x)*p), R2 = 0.999). Typical levels for endogenous TTR in the CSF of healthy individuals have been reported at 17 μg/mL.33 The expected clinical levels were shown to fall within the linear range of the calibration curve. SDS-PAGE Western Blotting for Qualitative Biomarker Analysis Comparison

Slab gel SDS-PAGE was performed on undiluted CSF and nasal discharge samples probing for TTR (Figure 4A). The SDSPAGE and Western blot process required approximately 8 h and resulted in a complex peak pattern. By visual inspection 3−4 bands were clearly seen in the CSF lane with the largest peak presumably albumin (66 kDa). The nasal mucus sample resulted in a minimum of 7 bands that were poorly resolved after SDS-PAGE. A profile plot of the SDS-PAGE pixel intensity (Figure 4B) allowed assessment of signal-to-noise ratio of the slab gel detection technique. Slab gel SDS-PAGE resulted in maximum 1260



Article

Figure 4. Western blot assays for TTR detection provide qualitative indication of CSF protein content in clinical samples. (A) Nonreducing slab gel SDS-PAGE of pooled CSF and nasal discharge samples performed in 4−12%T gel with subsequent Coomassie Blue staining requires ∼5 h. CSF has 4 major bands in profile while mucus has 7−8 bands. (B) SDS-PAGE gel signal intensity profile for undiluted CSF and nasal discharge. Maximum peak S/N ratios are approximately 128 and 187 for CSF and nasal mucus respectively, a 100-fold decrease from the on-chip assay S/N. Proteins are labeled with purported identity based on molecular weight (PGDSprostaglandin D synthase, TTRtransthyretin, HSAhuman serum albumin, β2TFbeta 2 transferrin, LYZlysozyme C, PIP, prolactin inducible protein, LNEClung and nasal epithelial carcinoma-associated protein, LF lactoferrin). (C) Coomassie staining of the clinical samples shows that the protein band profile for CSF differs significantly from nasal mucus in intensity and protein content with TTR tetramer (55 kDa) prevalent in CSF Coomassie stains while dimer (28 kDa) and monomer (14 kDa) isoforms are detected by Western blot. (D) Western blotting for TTR confirmed presence in 5 of 5 purported CSF samples (lanes 2, 5, 7, 10) and 2 of 5 purported mucus samples (lanes 8 and 9). Combined SDS-PAGE with subsequent Western blotting on clinical samples required ∼12 h.

samples from lanes 8 and 9 suggesting possible rhinorrhea or contamination with CSF. The on-chip method only detected TTR in the mucus sample from lane 8 but not lane 9. An additional discrepancy was seen in the control TTR purified protein sample which was detected via the on-chip method but not with the slab-gel Western blot method (Lane 1, Figure 4D); however different purified protein standards were used for the on chip vs Western blot assay thus affinity to the same detection Ab would be expected to vary. For the representative samples also analyzed via mass spectrometry, the TTR detection in both the Western blot and on-chip techniques correlated with mass spectrometry results.

3, 4, 6, 8, and 9 contained samples characterized as mucus during collection. The Coomassie stain demonstrated that all clinical samples, whether putative mucus or CSF, contained proteins of similar size to the TTR tetramer, however in the mucus bands there were several additional high concentration protein bands near 25, 70, and 200 kDa. The presence of multiple proteins in the 50−70 kDa size range made mobility-based identification ambiguous therefore Western blot for TTR antibody affinity was performed on the clinical samples resulting in the detection of TTR dimer in lanes 2, 5, and 7−10. Results showed that TTR antibody binding was primarily selective to the TTR dimer isoform (28 kDa). There was also detection of a smaller band around 15 kDa as well as several larger proteins (∼150 kDa) in several lanes possibly indicating presence of TTR monomer and larger mass isoforms or antibody aggregates. However, binding to these large and small bands was not considered for the qualitative analysis for which TTR dimer binding was used as positive confirmation of CSF. Table 2 shows a summary of the qualitative Western blot identification results compared to both on-chip TTR detection and LC/MS for available samples. All purported CSF samples showed detection of TTR via both Western blot and the on-chip method. Additionally, Western blot was able to detect TTR dimer in the purported mucus

ELISA Assays for Quantitative Biomarker Analysis Comparison

TTR ELISA assays were run as the benchtop method to compare biomarker detection capability in complex sample matrices with the on-chip method and slab gel Western blotting. Additionally, a β2TF ELISA was run as a benchmark since it is used in a commercially available immunofixation electrophoresis assay for CSF rhinorrhea detection. β2TF detection was not performed with on-chip immunosubtraction or Western blot due to the difficulty in separation of the bands from the high concentration of comigrating human serum albumin which 1261



Article

Table 2. Detection of TTR via Slab Gel Western Blot, OnChip Immunosubtraction, and Mass Spectrometry sample no.

preliminary I.D.

gel lane #

Western Blot (TTR)

on-chip (TTR)

control

purified TTR P1 CSF P1 mucus P2 mucus P3 CSF P3 mucus P4 CSF P8 mucus P10 mucus P12 CSF

1

−−

++

N/A

2 3 4 5 6 7 8 9 10

++ −− −− ++ −− ++ + + ++

++ −− −− ++ −− + + −− +

N/A N/A N/A N/A N/A N/A N/A N/A β2TF (+) TTR (+) PIP (−) β2TF (−) TTR (−) PIP (+) β2TF (+) TTR (+) PIP (−)

1 2 3 4 5 6 11 16 18

19

baseline mucus

*

−−

−−

20

baseline CSF

*

++

++

Table 4. Performance Characteristics for on-chip and ELISA methods compared to Western blot Benchtop Standard

LC/MS (TTR, β2TF, or PIP)

on-chip (TTR) (μg/mL)

1 4 6 18 20

P1 CSF P3 CSF P4 CSF P12 CSF CSF baseline

187.77 54.69 124.95 9.31 1256.42

no detection no detection 56.81 7.56 no detection

28.89 20.25 6.29 2.16 27

Western Blot ELISA (TTR) ELISA (β2-TF) on-chip immunosubtraction (TTR)

N/A 100 70 88

N/A 50 57 100

8−12 h 4 ha 6 ha 5 min

■

DISCUSSION Several putative biomarkers for stratifying CSF from nasal mucus have been identified previously including CSF specific proteins not found in mucus (i.e., transthyretin, beta-2transferrin, and L-PGDS) and, conversely, mucus specific proteins not found in CSF (i.e., prolactin-inducible protein).4 The on-chip detection of CSF relies on the quantitation of TTR, a serum and CSF carrier of thyroxine and retinol which is a readily detectable protein in high abundance in CSF, where it ranges from the second to seventh most prevalent CSF matrix protein based on shotgun proteomic analysis.34,35 Results of mass spectrometry run on baseline mucus, baseline CSF, and clinical CSF samples confirmed agreement with the literature on the presence and identity of stratifying biomarkers between CSF and mucus.4 In particular, transthyretin and beta-2transferrin were both found in high abundance in a baseline CSF sample while not present or found in low concentrations for the baseline mucus sample. Since post-translational modifications were not accounted for in the mass spectrometry analysis, the detection of transferrin in baseline mucus was not definitively the beta-2-transferrin isoform indicative of CSF contamination. PIP was an effective indicator of mucus as it was not present at any concentration in the baseline or clinical CSF samples. Results also demonstrated important differences between the baseline CSF sample obtained via lumbar puncture from healthy patients and the surgically obtained CSF rhinorrhea representative clinical sample. The relative decrease in CSF specific proteins (i.e., TTR, β2TF) and increase in serum proteins (i.e., haptoglobin, hemoglobin, etc.) suggested that visual inspection of apparently nonturbid samples was not sufficient to ensure nonsignificant blood contamination; which is of importance for spontaneous rhinorrhea, where the sample provided by the patient appears nonbloody. Contamination with blood has significant implications for usefulness of ELISA assays in particular as most are incompatible with hemolyzed samples since high concentration matrix proteins (e.g., albumin, IgG) can cause nonspecific interaction; while the high iron content of proteins such as hemoglobin strongly affect optical density measurements for automated plate readers where optical density measurements (550 nm) have previously been shown to increase linearly with hemoglobin concentration in brain tissue homogenate.36 The Western blot and on-chip immunosubtraction assays were more robust to small amounts of blood contamination since proteins were separated and captured via target antibody for less ambiguous qualitative

Table 3. Comparative Results of CSF Quantitation from Spontaneous Rhinorrhea Representative Clinical Samples by ELISA and On-Chip Immunosubtraction ELISA B2TF (μg/mL)

assay time

β2TF while the on-chip assay specificity was 100%. The TTR ELISA had 100% sensitivity, while the β2TF ELISA had 70%, compared to 88% sensitivity for the on-chip immunosubtraction method.

forms 67% of total CSF protein.33 Samples were separated and preidentified as purported CSF, mucus, or maxillary sinus fluid based on their visual characterization and collection location during surgery. Grossly hemolyzed samples were excluded from the study as both ELISA protocols indicated noncompatibility with hemolyzed samples. Additionally, mucus samples were excluded from ELISA analysis since preliminary ELISA results indicated that significant nonspecific binding occurred due to mucus matrix proteins (see Supporting Information S.3). Results of the TTR ELISA on CSF samples indicated that 5 of 5 purported CSF clinical samples tested positive for TTR at varying concentrations ranging from 9 to 1256 μg/mL (Table 3).

ELISA TTR (μg/mL)

specificity (%)

Direct competitive ELISA required 4 h while indirect sandwich ELISA required 6 h.

Not shown; (+) positive detection, (−) no detection.

initial I.D.

sensitivity (%)

a

*

sample no.

method

β2TF was only detectable in 2 of 5 purported CSF clinical samples at concentrations of 7.5 and 56 μg/mL. The on-chip quantitation technique detected TTR concentrations in all tested samples ranging between 2 and 28 μg/mL. Assay sensitivity and specificity was calculated for both ELISA assays as well as on-chip immunosubtraction compared to Western blotting which was used as the gold-standard for TTR detection (Table 4). All nonturbid spontaneous rhinorrhea representative samples including clinical CSF, baseline CSF, and mucus comprised the total pool of 11 samples analyzed (see Supporting Information S.3 for ELISA detection of mucus). Results showed that the ELISA assays had low specificity for CSF detection at 50% and 57%, respectively, for TTR and 1262



Article

TTR ≈ pH 8.3, β2TF ≈ pH 7.4) and promoting nonspecific charge-based interaction with both the target antibodies and coexisting matrix proteins as previously observed in the literature;29,37 and which was also supported by our ELISA nonspecific binding investigation (see Supporting Information S.3). Given the high sensitivity but slow assay time for ELISA, the technique could provide a useful CSF rhinorrhea confirmatory diagnostic assay. However, assay development would likely require significant sample preparation and optimization of the buffer systems to accommodate alkaline mucus matrix proteins, which makes adaptation of the method unlikely for emergency medicine or point-of-care CSF analysis. The on-chip immunosubtraction method proved most amenable to the CSF biomarker proteomic study given the high specificity and high-throughput compared to benchtop techniques. The advantage of the on-chip method was the short assay time (5−10 min) compared to ELISA (4−6 h) or slab gel Western blot (5−12 h). On-chip detection was limited to samples not containing blood since the abundance of serum matrix proteins saturated on-chip readout (serum total protein concentration ∼200× that of CSF). Given that spontaneous rhinorrhea typically requires the stratification between clear mucus and CSF, the on-chip method was useful for spontaneous rhinorrhea diagnosis as opposed to intraoperative or postoperative trauma induced rhinorrhea detection. The qualitative nature of the Western blot detection also proved a more appropriate application for which to develop a rapid diagnostic or proteomic tool to assess samples since the CSF rhinorrhea screening assay would seek a yes/no diagnosis of leakage. As seen from the quantitative results in Table 3, qualitative on-chip measurements can be made with higher confidence than quantitative measurements which were prone to fluctuation resulting from nonstandardized operating room sample collection methods (i.e., nonstandardized nasal lavage fluid wash procedure) which made determining accurate cutoff levels for yes/no diagnosis difficult.

detection. The total number of proteins detected in the clinical CSF (207) was also significantly more than that observed in baseline CSF (103) as previously reported in the literature.10 This points to contamination of the apparently nonturbid clinical CSF samples with blood specific proteins potentially affecting their ELISA results; however, some of the additional clinical CSF proteins may also originate from patient specific pathologies as some sample collection was done in conjunction with surgical procedures for tumor removal. On-chip separation of CSF and mucus samples resulted in identification of only the major peaks in each sample (CSF2 peaks, mucus4 peaks) within the 1.5 mm separation distance. The signal-to-noise ratio on-chip was improved ∼100-fold over the slab gel SDS-PAGE in an assay run in 1/30th of the time and 1/10th the separation distance resulting from the sample stacking effect due to the abrupt change in mobility of analytes migrating across a discrete large to small pore-size interface as previously described for PA sieving gels.21 However, several minor peaks present in the slab gel separation (CSF4 peaks, mucus6 peaks) appeared to be attenuated or undetected onchip; likely due to them comigrating with other matrix proteins under native separation conditions or being larger than the immunofilter molecular weight cutoff at the start of the separation channel. Given that the biomarker of interest, TTR, did not fall within the range of attenuated proteins and was detectable with improved S/N with the on-chip method compared to slab gel; the reduction of nontarget peaks present on-chip facilitated identification and tracking of the target peaks during quantitation when compared to slab gel SDS-PAGE due to the increased separation resolution. Accurate TTR peak identification (88% sensitivity, 100% specificity) was also facilitated on-chip by performing native PAGE to separate proteins based on charge-to-mass ratio since several mucus and serum matrix proteins migrated with similar mobilities during SDS-PAGE due to their comparable size. TTR Western blots demonstrated relative insensitivity to blood contamination, but a significant drawback impacting throughput and diagnostic application was the need to optimize membrane blocking procedures to reduce nonspecific background signal for the chosen TTR primary antibody (see Supporting Information Figure S-4). A polyclonal anti-TTR primary antibody was used for Western blotting for which some cross-reactivity with other matrix proteins would be expected; however, by limiting positive identification of TTR to the presence of the dimer band the Western blot results were consistent with mass spectrometry and suitable for use as the comparative benchtop gold standard measurement for TTR. Previous research corroborates findings that during SDS-PAGE TTR can be expected to migrate predominantly in the 14 kDa monomer, with a lower concentration present in the 28 kDa dimer, and the lowest concentration present in the 55 kDa native tetramer.10 Significant limitations to CSF rhinorrhea diagnostic capability were shown for both the TTR and β2TF ELISAs as evidenced by the low specificity for these methods (50% and 57%, respectively). ELISA results were consistently higher than the TTR concentrations detected on-chip reaching 100× the expected clinical concentrations for detection of the baseline CSF sample. In particular, this was a direct result of high abundance alkaline matrix proteins in mucus including lactoferrin (pI 8.7) and lysozyme C (pI 9.28) obtaining positive charge under ELISA conditions (ELISA wash buffers:

■

CONCLUSIONS A microfluidic device was introduced for validating TTR biomarker detection in cerebrospinal fluid rhinorrhea clinical samples. While CSF rhinorrhea is a serious condition, current state-of-the-art diagnostics are expensive or slow (MRI, CT scan, or ELISA). We showed the capability of a microfluidic immunoassay to accept and processes raw biological fluids from nasal leakage prior to rapidly characterizing biomarker content via immunosubtraction PAGE. Miniaturizing the assay onto a microfluidic chip with integrated sample preparation offered advantages for detecting CSF rhinorrhea including increased automation, portability, multiplexed detection of biomarker panels (PIP, TTR, and HSA) and reduced assay time from 5 to 12 h for slab gel SDS-PAGE with Western blot to 5−10 min for on-chip analysis. Results demonstrated the ability to rapidly identify and differentiate CSF from nasal mucus based on TTR detection. Comparison to benchtop methods showed that the on-chip technique had better specificity and less susceptibility to nonspecific matrix protein interference than ELISA while quantifying TTR concentration which is not possible with slab gel Western blot. Although the on-chip method was limited to detection of nonhemolyzed samples representative of spontaneous rhinorrhea (i.e., not containing blood), on-chip analysis was more robust to small amounts of blood found in clinical 1263


Journal of Proteome Research samples than the ELISA assays. Results point to the efficacy of the on-chip technique for CSF rhinorrhea screening diagnosis and monitoring in clinical laboratories and for emergency medicine to promote more widespread implementation of effective screening protocols while reducing cost and assay time to result. The demonstrated on-chip format also provides a means for performing rapid and high-throughput biomarker validation studies from low-volume patient samples not amenable to ELISA. Given the presence of TTR in blood that inhibits the use of TTR as a diagnostic biomarker in blood contaminated CSF, future efforts are underway to incorporate the on-chip technique for detecting β2TF as an additional panel diagnostic biomarker for spontaneous rhinorrhea detection.

■

ACKNOWLEDGMENTS

■

REFERENCES

The authors acknowledge Dr. Manish K. Aghi for collection of additional samples and Dr. Lori Kohlstaedt of the QB3 Vincent Coates Proteomics/Mass Spectrometry Lab for guidance with mass spectrometry data analysis.

(1) Goyal, V.; Srinivasan, M. J. Gen. Intern. Med. 2011, 26 (3), 345. (2) McMains, K. C.; Gross, C. W.; Kountakis, S. E. Laryngoscope 2004, 114, 1833−1837. (3) Schlosser, R. J.; Bolger, W. E. Laryngoscope 2004, 114, 255−265. (4) Burkhard, P. R.; Rodrigo, N.; May, D.; Sztajzel, R.; Sanchez, J. C.; Hochstrasser, D. F.; Shiffer, E.; Reverdin, A.; Lacroix, J. S. Electrophoresis 2001, 22, 1826−1833. (5) Meco, C.; Oberascher, G. Laryngoscope 2004, 114, 991−999. (6) Schnabel, C.; Di Martino, E.; Gilsbach, J. M.; Riediger, D.; Gressner, A. M.; Kunz, D. Clin. Chem. 2004, 50, 661−663. (7) Bateman, N.; Jones, N. S. J. Laryngol. Otol. 2000, 114, 462−464. (8) Keerl, R.; Weber, R. K.; Draf, W.; Wienke, A.; Schaefer, S. D. Laryngoscope 2004, 114, 266−272. (9) Warnecke, A.; Averbeck, T.; Wurster, U.; Harmening, M.; Lenarz, T.; Stover, T. Arch. Otolaryngol.-Head Neck Surg. 2004, 130, 1178− 1184. (10) Marchi, N.; Fazio, V.; Cucullo, L.; Kight, K.; Masaryk, T.; Barnett, G.; Volgelbaum, M.; Kinter, M.; Rasmussen, P.; Mayberg, M. R.; Janigro, D. J. Neurosci. 2003, 23, 1949−1955. (11) Gallo, P.; Bracco, F.; Morara, S.; Battistin, L.; Tavolato, B. J. Neurol. Sci. 1985, 70, 81−92. (12) Van Landeghem, G. F.; D’Haese, P. C.; Lamberts, L. V.; De Broe, M. E. Anal. Bioanal. Chem. 1996, 355, 96−97. (13) Bossuyt, X.; Bogaerts, A.; Schiettekatte, G.; Blanckaert, N. Clin. Chem. 1998, 44, 760−764. (14) Paquette, D. M.; Banks, P. R. Electrophoresis 2001, 22, 2391− 2397. (15) Caton, J. E.; Holladay, D. W.; Anderson, N. G. Abs. Papers Am. Chem. Soc. 1973, 31−. (16) Keren, D. F. Arch. Pathol. Lab. Med. 1999, 123, 126−132. (17) Keren, D. F. Protein Electrophoresis in Clinical Diagnostics; Arnold Publishers: London, 2003. (18) Apori, A. A.; Herr, A. E. In Clinical Applications of Capillary Electrophoresis; Phillips, T. M., Kalish, H., Eds.; Humana Press: New York, 2012. (19) Apori, A. A.; Herr, A. E. Anal. Chem. 2011, 83, 2691−2698. (20) Karns, K.; Herr, A. E. Bioanalysis 2011, 3 (19), 2161−2165. (21) Hou, C. L.; Herr, A. E. Anal. Chem. 2010, 82, 3343−3351. (22) Thongboonkerd, V.; Songtawee, N.; Sritippayawan, S. J. Proteome Res. 2007, 6, 2011−2018. (23) Nwosu, C.; Aldredge, D.; Hyeyoung, L.; Lerno, L.; Zivkovic, A.; German, J.; Lebrilla, C. J. Proteome Res. 2012, 11 (5), 2912−2924. (24) He, M.; Herr, A. E. J. Am. Chem. Soc. 2010, 132, 2512−2513. (25) Herr, A. E.; Throckmorton, D. J.; Davenport, A. A.; Singh, A. K. Anal. Chem. 2005, 77, 585−590. (26) Yue, G. E.; Roper, M. G.; Balchunas, C.; Pulsipher, A.; Coon, J. J.; Shabanowitz, J.; Hunt, D. F.; Landers, J. P.; Ferrance, J. P. Anal. Chim. Acta 2006, 564, 116−122. (27) Phillips, T. M.; Wellner, E. J. Chromatogr. A 2006, 1111, 106− 111. (28) Herr, A. E.; Hatch, A. V.; Throckmorton, D. J.; Tran, H. M.; Brennan, J. S.; Giannobile, W. V.; Singh, A. K. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5268−5273. (29) Karns, K.; Herr, A. E. Anal. Chem. 2011, 83 (21), 8115−8122. (30) Apori, A. A.; and Herr, A. E. In The 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences (uTAS2010); Chemical and Biological Microsystems Society: Groningen, Netherlands, 2010, pp 2056−2058. (31) Phillips, T. M. Electrophoresis 2004, 25, 1652−1659. (32) Palfrey, S. M. Methods Mol. Med. 1999, 27, 39−46.

ASSOCIATED CONTENT

* Supporting Information S

S.1 On-chip immunosubtraction with integrated sample preparation; raw sample preparation integrated with immunosubtraction PAGE (Figure S-1); homogenous assay enables immunosubtraction of putative protein biomarkers from complex biological fluids and as multi-analyte panels (Figure S-2); S.2 Western blot hemolyzed sample and background binding analysis; SDS-PAGE and Western blot for hemolyzed samples (Figure S-3); hemolyzed sample list for Western blotting (Table S-1); optimization of PVDF membrane blocking for TTR Western blot probing (Figure S-4); S.3 ELISA protocol and non-specific signal investigation; measured pH for ELISA and clinical samples (Table S-2); reported isoelectric point (pI) for abundant proteins in clinical samples (Table S-3); ELISA non-specific binding results (Table S-4); S.4 mass spectrometry results; Sequest parameter modifications (Table S-5); baseline CSF mass spectrometry results (Table S-6); baseline mucus mass spectrometry results (Table S-7); and clinical CSF mass spectrometry results (Table S-8). Additional supporting files include the SEQUEST search parameters and raw mass spectrometry results. This material is available free of charge via the Internet at http://pubs.acs.org

■

■

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

A.A.A. and A.E.H. designed research, analyzed data, and wrote the paper. A.A.A. performed on-chip experiments. A.A.A. and M.N.B. performed benchtop experiments. I.H.E. collected patient clinical samples during surgery. All authors have given approval to the final version of the manuscript. Funding

This material is based upon work supported by the Defense Advanced Research Projects Agency (DARPA) under Award N66001−09−1−2118 and the National Institutes of Health through the NIH Director’s New Innovator Award Program (AEH, 1DP2OD007294). A.A.A. is a National Defense Science and Engineering Graduate (NDSEG) Fellow and UNCF/ Merck Fellow. A.E.H. is an Alfred P. Sloan Foundation Research Fellow in chemistry. Notes

The authors declare no competing financial interest. 1264



Article

(33) Irani, D. Cerebrospinal Fluid in Clinical Practice; Saunders Elsevier: Philadelphia, 2009. (34) Waller, L. N.; Shores, K.; Knapp, D. R. J. Proteome Res. 2008, 7, 4577−4584. (35) Shores, K. S.; Knapp, D. R. J. Proteome Res. 2007, 6, 3739−3751. (36) Choudhri, T. F.; Hoh, B. L.; Solomon, R. A.; Connolly, E. S.; Pinsky, D. J. Stroke 1997, 28, 2296−2302. (37) Hekman, A. Biochim. Biophys. Acta 1971, 251, 380−387.

1265


Microfluidic Validation of Diagnostic Protein Markers for Spontaneous

Recommend Documents