Name: hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins
Uploaded: 2014-08-07T20:45:00.000+00:00
Duration: 10 min 5 s
Description: Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

Michael Henry, Dublin City University, kindly assisted with the data collection and analysis shown in Figure 5. This work was supported partially by (1) George Mason University, (2) the Italian IstitutoSuperiore di Sanita&#8217; in the framework of the Italy/USA cooperation agreement between the U.S. Department of Health and Human Services, George Mason University, and the Italian Ministry of Public Health, (3) NIH, IMAT program grants 1R21CA137706-01 and 1R33CA173359-01 to LAL, and (4) Ceres Nanosciences, Inc.

Human plasma and urine was collected from healthy volunteer donors, with written informed consent, following George Mason University Institutional Review Board approved protocols. Donors were equally distributed between Caucasian males and females between the ages of 25 and 42. Samples were analyzed individually and were not pooled.
1. Nanoparticle Processing of Serum or Plasma Samples
Potential low abundant biomarkers in plasma are captured, in solution, with hydrogel nanoparticles. The particles are added to the plasma, incubated, separated by centrifugation, washed, and the captured proteins are eluted. The eluted proteins are dried under nitrogen flow for downstream mass spectrometry sequencing and identification.
<ol>
	<li>Dilute 500 &#181;l of serum 1:2 with 50 mM TrisHCl pH 7 (500 &#181;l serum + 500 &#181;l TrisHCl) in a microcentrifuge tube.</li>
	<li>Add 500 &#181;L of poly(NIPAm/AAc) core nanoparticles. Incubate for 15 min at RT.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge equipped with a fixed-angle rotor. Remove and discard the supernatant.</li>
	<li>Add 500 &#181;l sodium thiocyanate (25 mM) to the pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge with a fixed angle rotor. Remove and discard the supernatant.</li>
	<li>Add 500 &#181;l of MilliQ water to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge with a fixed angle rotor. Remove and discard the supernatant.</li>
	<li>Prepare fresh elution buffer: Add 700 &#181;l acetonitrile to 300 &#181;l ammonium hydroxide in a clean tube. Caution: Ammonium hydroxide is corrosive. Use with appropriate ventilation. Note: The elution buffer must be prepared immediately before use. Do not store the elution buffer.</li>
	<li>Add 300 &#181;l of elution buffer to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Incubate for 15 min at RT.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge with a fixed angle rotor. Remove and save the eluate in a clean, labeled microcentrifuge tube.</li>
	<li>Repeat steps 1.9 &#8211; 1.10. Combine the two eluates into one microcentrifuge tube.</li>
	<li>Dry the eluates under nitrogen flow in a nitrogen evaporator manifold at 42.1&#160;&#176;C, with air flow set to 8 (Figure 2F).</li>
	<li>Store the dried eluate at RT for O/N storage, or at -20&#160;&#176;C for long term storage, prior to mass spectrometry, western blotting, or ELISA assays (optional).</li>
</ol>
2. Nanoparticle Processing of Urine Samples
Normal urine contains less than 30 mg/dl protein and less than 1+ blood. However, many diseases/conditions may alter the normal levels of urine protein and blood. To aid in determining the optimal volume of nanoparticles to add to the urine sample, a urinalysis is performed prior to nanoparticle harvesting. Urine biomarkers may exist in extremely low concentrations, which may require optimizing the ratio of nanoparticles to urine volume. This procedure describes nanoparticle harvesting of urine samples for downstream western blot analysis.
<ol>
	<li>Collect urine samples in a clean, dry plastic cup. A 22 ml minimum volume is required. Store urine specimens at -80 &#176;C until ready to analyze.</li>
	<li>Thaw the frozen urine at RT or at 4 &#176;C O/N. Mix briefly on a vortex mixer. Pour at least 22 ml of urine into a 50 ml conical bottom polypropylene tube.</li>
	<li>Spin the urine in a centrifuge with a swing-out rotor at 3,700 x g for 15 min. Decant the urine, without disturbing the pellet, into a clean 50 ml conical bottom polypropylene tube. Discard the pellet.</li>
	<li>Perform urinalysis using a multi-analyte &#8220;urine dipstick&#8221; reagent strip. Note: store the reagent strips in a tightly closed container away from direct sunlight and humidity17, 18.
	<ol>
		<li>Lay the reagent strip, face-up, on a clean, dry paper towel.</li>
		<li>Use a disposable pipette to aspirate 1 ml of the urine prepared in step 2.3.</li>
		<li>Set a timer for 2 min but do not start it. Quickly dispense 1 - 2 drops of urine on each test pad of the reagent strip. Immediately start the timer. Note: Do not submerge the reagent strip directly in the urine container. The dye/chemical indicators in the reagent strip can potentially leach out of the reagent strip into the urine container.</li>
		<li>At the time indicated on the reagent strip container, record the qualitative results for the various analytes on the reagent strip by comparing the color of the individual reagent strips to the corresponding color-coded indicators on the reagent container. Typical normal urine results are: (normal or negative) for blood, protein, leukocytes, nitrite, glucose, ketone, bilirubin, and urobilinogen; Urine pH (5.5 - 7.0); specific gravity (1.001 - 1.020). Note: High protein levels in a urine specimen may compete with the protein of interest for binding sites on the nanoparticles. To maximize protein harvesting with nanoparticles, the nanoparticle volume/urine volume ratio can be adjusted to either limit competition from high abundance proteins, or can be optimized to harvest proteins that exist in very low concentrations. If the urine protein value is 1+ or greater, add 2x volumes of nanoparticles in step 2.5 below. If the analyte of interest is a very low abundant protein, it may be necessary to increase the volume of nanoparticles up to 2 ml&#160;(Figure 3).</li>
	</ol>
	</li>
	<li>Transfer 20 ml of the clarified urine from step 2.3 into a clean 50 ml conical bottom polypropylene tube. Do not disturb any debris/pellet that may be in the bottom of the urine tube. Add 200 &#181;l of nanoparticles to the 20 ml urine sample. Mix briefly on a vortex mixer. Note: If the urine protein value is 1+ or greater, add 400 &#181;l of nanoparticles to 20 ml urine.</li>
	<li>Incubate the urine/nanoparticle mixture for 30 min at RT, without rocking/mixing.</li>
	<li>Spin the urine/nanoparticle suspension in a centrifuge equipped with a swing-out rotor at 3,700 x g for 10 min. Note: If a pellet is not visible following centrifugation, spin the samples for an additional 2 &#8211; 7 min.</li>
	<li>Remove and discard the supernatant. Add 500 &#181;l MilliQ water to the nanoparticle pellet.</li>
	<li>Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Transfer the nanoparticle solution to a clean 1.5 ml microcentrifuge tube.</li>
	<li>Spin the nanoparticles in a centrifuge equipped with a fixed-angle rotor at 16,100 x g for 10 min. Note: If a pellet is not visible following centrifugation, spin the samples for an additional 2 &#8211; 7 min.</li>
	<li>Remove and discard the supernatant.</li>
	<li>Repeat steps 2.8 and 2.11 (twice) with 200 &#181;l 18 MilliQ water to wash the nanoparticles.</li>
	<li>Prepare fresh elution buffer: Add 970 &#181;l acetonitrile to 30 &#181;l ammonium hydroxide in a clean tube. Caution: Ammonium hydroxide is corrosive. Use with appropriate ventilation. Note: The elution buffer must be prepared immediately before use. Do not store the elution buffer.</li>
	<li>Add 20 &#956;l elution buffer to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Incubate for 15 min at RT.</li>
	<li>Spin the nanoparticle samples at 16,100 x g for 10 min. Remove and save the supernatant in a clean, 1.5 ml microcentrifuge tube. Do not disrupt the nanoparticle pellet. Discard the pellet in the biohazard trash.</li>
	<li>Place the samples in a rack in a chemical fume hood. Open the caps and incubate at RT for 30 min. Alternatively, place the samples under nitrogen flow at 40 &#176;C until dry (1 - 2 hr).</li>
	<li>Add 15 &#181;l Tris-glycine SDS sample buffer (2x) to the samples. Heat at 100 &#176;C in a dry heat block for 5 min with the caps open or until the volume left in the tube is no more than 20 &#181;l. Note: do not use a boiling water bath. The humidity from the water bath will prevent evaporation of the buffer.</li>
	<li>Place the cap on the tubes. Remove the tubes from the heat block. The sample can be stored at -80 &#176;C or used immediately for downstream western blot analysis.</li>
</ol>

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Benjamin Espina is an employee of Ceres Nanosciences, Inc. that produces reagents used in this Article. Lance Liotta, Alessandra Luchini and Virginia Espina hold patents (US patent 7,935,518 and/or 8,497,137) on the nanoparticle technology used in this Article. As university employees they are entitled to receive royalty from these patents per university policy. Lance Liotta and Alessandra Luchini are shareholders in Ceres Nanosciences and serve on the Scientific Advisory Board.

Clinical Relevance
A serum or plasma sample is thought to contain low-abundance circulating proteins and peptides which can provide a rich source of information regarding the state of the organism as a whole. Despite the promise of serum proteomics, there are three fundamental and serious physiologic barriers thwarting biomarker discovery and translation to clinical benefit10, 11, 16, 25.
1. Important diagnostic biomarkers may exist in extremely low-abundance (concentration) in blood. Early stage diseased tissue, such as pre-metastatic cancer lesions, may constitute less than a few mm3. Biomarkers shed into the circulation from such a small tissue volume will become highly diluted in the entire blood volume. Relevant analytes may exist below the detection limits of mass spectrometry and conventional immunoassays.
2. Low abundance biomarkers/proteins are masked by the presence of high abundant proteins such as albumin and immunoglobulin, which represent up to 90% of the plasma proteome14.
3. Proteins and peptides are susceptible to degradation by endogenous and exogenous proteinases following venipuncture and sample transport/storage. Protein degradation can lead to false positive or false negative results35.
Hydrogel nanoparticles were developed as porous, buoyant, polymers containing anthraquinonetriazine dyes and/or vinylsulfonic acid shells for protein and peptide harvesting and preservation in body fluids11, 13. Our group synthesized and tested hydrogel nanoparticles functionalized with a plethora of organic dyes (i.e., sulfonated and non-sulfonatedanthraquinonetriazine dyes) and showed the preferential affinities for several protein analytes11, 13. We also established protocols for biomarker discovery that use hydrogel nanoparticles with lymph, saliva, cerebrospinal fluid, sweat, urine, plasma, blood, or serum specimens11, 13, 23, 24, 26.
Optimizing the nanoparticle harvesting parameters prior to harvesting proteins from precious clinical/research specimens is necessary for ideal results. The amount of protein in the samples should be quantified to determine the optimum ratio of nanoparticles to sample volume. For analytes of unknown concentration, a dose-response curve using recombinant proteins provides information regarding the limits of detection. If there is adequate sample, various types of nanoparticles can be used to determine the ideal nanoparticle for the analyte and specimen.
Urinalysis prior to nanoparticle harvesting provides a urine sample quality control check. The affinity of the nanoparticle dye bait depends on the isoelectric point of the protein of interest and the pH of the surrounding medium. Urine pH between 5.5 and 7.0 is optimal for protein harvesting with the poly(NIPAm/dye) nanoparticles. The presence of hemolyzed or intact red blood cells (1+ blood on the reagent strip) does not interfere with nanoparticle harvesting.
In this protocol we focused on the application of the hydrogel nanoparticles to harvest proteins from plasma and urine. Future applications could comprise other body fluids such as vitreous of the eye, or synovial fluid. Potential in vivo applications can be envisioned for the future in which nanoparticles are injected into diseased tissue to collect biomolecules. Future work will also be focused on the development of new devices for the capture and preservation of biomarkers, such as a nanoparticle-based skin patch for the analysis of the skin transudate proteome.

Despite the completion of the human genome sequencing, significant progress has not been made in identifying biomarkers predictive of early stage disease, or that correlate with therapeutic outcome, or prognosis1. One reason for this lack of progress is that many potentially significant biomarkers exist at a concentration below the detection limit of conventional mass spectrometry and other biomarker discovery platforms. Mass spectrometry (MS) and Multiple Reaction Monitoring (MRM) have a detection sensitivity typically greater than 50 ng/ml while the majority of the analytes measured by immunoassays in a clinical laboratory fall in the range between 50 pg/ml and 10 ng/ml. This means that many biomarkers, particularly in the early stage of a disease cannot be detected by conventional MS and MRM2. In addition the presence of high-abundance proteins such as albumin and immunoglobulin in complex biological fluids often mask by billion-fold excess low-abundance, low molecular weight proteins and peptides3, 4. For this reason several sample preparatory steps are required prior to mass spectrometry sequencing and identification. One such preparatory step employs the depletion of high-abundance proteins with commercially available depletion columns5-8. Unfortunately this step leads to the reduction of the yield of candidate biomarkers because they are often non-covalently associated with carrier proteins that are being removed. Another challenge is represented by the stability of candidate biomarkers ex-vivo once the samples are collected. Proteins are subject to degradation by endogenous or exogenous proteases9. Hydrogel nanoparticles can transcend these critical challenges by amplifying the putative biomarker concentration to a level within the range of the assay, while protecting the protein from degradation10-13.
It&#8217;s important to note that LMW proteins in blood are a mixture of small intact proteins as well as fragments of large proteins. Tissue-derived proteins larger than 60 kDa are too large to passively enter the blood stream through the vascular basement membrane, but they can be represented in blood as peptides or protein fragments14. Our goal is to measure novel circulating biomarkers that can be candidates for early detection of disease, patient stratification for therapy, and monitoring the response to therapy. Our nanoparticles are created to selectively exclude high abundance immunoglobulins and albumin, while simultaneously capturing smaller proteins and peptides and concentrating them up to 100-fold depending on the starting volume.
Our group identified a set of small organic dyes which can successfully act as high affinity molecular baits for proteins and peptides. Protein-dye binding is thought to be due to a combination of hydrophobic and electrostatic interactions. The aromatic rings on the dye interleave with proteins via hydrophobic pockets on the protein surface11.
The baits, depending on their chemistry, show a particular affinity for selected classes of analytes. The baits compete with the carrier proteins, such as albumin, for the proteins or peptides. The low-molecular weight proteins/peptides become trapped in the particle. High-molecular weight proteins such as albumin and immunoglobulin are prevented from entering the particle because of the sieving capability due to the restrictive pore of the hydrogel11(Figure 1).
Hydrogel nanoparticles are synthesized by precipitation polymerization initiated by ammonium persulfate11. N-isopropylacrylamide (NIPAm), co-monomers of acrylic acid (AAc) and allylamine (AA) and cross-linker N,N&#8217;-Methylenebisacrylamide (BIS) are allowed to react at 70 &#176;C for 6 hr in dilute conditions11, 13. The high protein binding affinity of poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAm-co-AAc) nanoparticlesis achieved by covalently incorporating amino-containing dyes (i.e., sulfonatedanthraquinonetriazine dyes) into the nanoparticles through an amidation reaction performed in aqueous or organic solvents depending on the hydrophilic/hydrophobic characteristics of the dyes11, 13. Nucleophilic substitution of the amine groups in the nanoparticle with the chloride atom of an anthraquinonetriazine dye is utilized to create dye-containing poly(NIPAm-co-Allylamine) (AA) nanoparticles11, 12. A two-step polymerization process is utilized to create hydrogel nanoparticles containing an outer shell of vinylsulfonic acid (VSA)11, 13.
Hydrogel nanoparticles can be applied to various biological fluids, including whole blood, plasma, serum, cerebrospinal fluid, sweat, and urine. In one step, in solution, the nanoparticles perform a rapid (within minutes) sequestration and concentration of low molecular weight analytes10, 11, 13, 15-18. Proteins are subsequently eluted from the nanoparticles and detected using western blotting19-21, mass spectrometry10, 11, 13, 15, 18, 22, 23, immunoassays/ELISA10, 11, 15, 18, or reverse phase protein microarray16, 24 assays. Nanoparticles functionalized with chemical bait, and presenting a core or core shell architecture, capture and concentrate proteins based on the bait/shell physicochemical properties. Different dyes incorporated into the nanoparticles will therefore capture different subsets of proteins with varying efficiency based on the dye affinity, pH of the solution, and the presence/absence of competing high-abundant proteins13. Furthermore, the quantity of nanoparticles in relation to the volume of the solution will affect the protein yield from the nanoparticles. These aspects of hydrogel nanoparticle harvesting are demonstrated using three different nanoparticle baits for harvesting proteins from plasma samples which contain high amounts of protein, and from urine samples which typically do not contain large amounts of protein. In this protocol we demonstrate harvesting and concentrating tumor necrosis factor alpha (TNF&#945;) from plasma samples using poly(NIPAm-co-AAc), Poly(NIPAm/dye), and core- shell nanoparticles (Poly(NIPAm-co-VSA)). Poly(NIPAm/dye) nanoparticles are shown to concentrate Mycobacterium species antigen that was added to human urine samples, to mimic Mycobacterium tuberculosis infected individuals.

<table><tbody><tr><td>Hydrogel nanoparticles</td><td>Ceres Nanoscience</td><td>CS003</td><td>NanoTrap ESP particles</td></tr><tr><td>18 M&amp;Omega;-cm water</td><td></td><td></td><td>Type 1 reagent grade water</td></tr><tr><td>Tris HCl, 50 mM pH 7.0</td><td>VWR</td><td>IC816116</td><td>50 mM, pH 7</td></tr><tr><td>Acetonitrile</td><td>BDH</td><td>BDH1103-4LP</td><td>Available from VWR</td></tr><tr><td>Ammonium Hydroxide NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>BDH</td><td>BDH3014</td><td>Available from VWR, assayed at 28-30% NH&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium thiocyanate 25 mM</td><td>Acros Organics</td><td>419675000</td><td>For serum/plasma samples</td></tr><tr><td>Multi-analyte Urine Reagent Strips</td><td>Siemens</td><td>2161</td><td>For urine samples</td></tr><tr><td>Tris-Glycine SDS Sample Buffer (2X)</td><td>Life Technologies</td><td>LC2676</td><td>Use at RT to prevent SDS from precipitating</td></tr><tr><td>Dry bath incubator (100 &lt;sup&gt;o&lt;/sup&gt;C) with heating block</td><td>Barnstead</td><td>11-715-125DQ</td><td>Do not substitute a boiling water bath</td></tr><tr><td>Nitrogen evaporator manifold</td><td>Organomation Associates</td><td>Microvap118</td><td>For serum/plasma samples</td></tr><tr><td>Centrifuge, swing-out rotor</td><td>Sorvall</td><td>Legend series</td><td>50 ml tube capacity, rcf 3,700 x g</td></tr><tr><td>Centrifuge, fixed angle rotor</td><td>Eppendorf</td><td>5424</td><td>1.7 ml microcentrifuge capacity, rcf 16,000 x g</td></tr><tr><td>50 ml conical centrifuge&amp;nbsp;tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td>With screw caps for urine samples</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>50-949-755</td><td></td></tr><tr><td>Timer</td><td>Fisher Scientific</td><td>S04782</td><td>Seconds/minutes</td></tr></tbody></table>

hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

hydrogel-nanoparticle-harvesting-plasma-or-urine-for-detecting-low

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.8K Views.  George Mason University. Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Video: Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Biochemistry

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Chemistry

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

Engineering

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes have the potential to serve as biomarkers for disease diagnosis and to monitor disease progression and treatment response. However, most exosome analysis procedures require exosome isolation and purification steps, which are usually time-consuming and labor-intensive, and thus of limited utility in clinical settings. This report describes a rapid procedure to analyze specific biomarkers on the outer membrane of exosomes without requiring separate isolation and purification steps. In this method, exosomes are captured on the surface of a slide by exosome-specific antibodies and then hybridized with nanoparticle-conjugated antibody probes specific to a disease. After hybridization, the abundance of the target exosome population is determined by analyzing low-magnification dark-field microscope (LMDFM) images of the bound nanoparticles. This approach can be easily adopted for research and clinical use to analyze membrane-associated exosome biomarkers linked to disease.

Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples.

Infected or malignant cells frequently secrete more exosomes, leading to elevated levels of disease-associated exosomes in the circulation. These exosomes ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Biology

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...