This work was supported by the National Institutes of Health [1DP2CA280622-01] and funding from Biolocity. We thank Dr. Maria Jennings and Andrea Fox for providing the HsADA1 and HsKYNase expression vectors.

1. Serum incubation
<ol>
	<li>Prepare a 10x stock of HsADA1 in 1x PBS pH 7.4 (1x PBS) at a final concentration of 10 &#181;M. Thaw a 15 mL aliquot of pooled human serum and pre-warm it to 37 &#176;C. Prewarm a 50 mL aliquot of 1x PBS to 37 &#176;C.</li>
	<li>Prepare the enzyme-serum incubation mixtures by adding 100 &#181;L of the 10x HsADA1 stock to 900 &#181;L of pooled human serum in a low-bind microcentrifuge tube, referred to as the enzyme + serum mixture. In a separate low-bind microcentrifuge tube, add 100 &#181;L of the 10x HsADA1 stock to 900 &#181;L of 1x PBS for comparison to a non-serum control, referred to as the enzyme + 1x PBS mixture. The final concentration of HsADA1 in each mixture will be 1 &#181;M.</li>
	<li>Prepare an additional control in a low-bind microcentrifuge tube consisting of 100 &#181;L of 1x PBS mixed with 900 &#181;L of pooled human serum. This will serve as the non-enzyme control for the enzyme + serum mixture. Additionally, make a control consisting of 1 mL of 1x PBS. This will serve as the non-enzyme control for the enzyme + 1x PBS mixture.</li>
	<li>Aliquot 100 &#181;L of each mixture from step 1.2 into separate low-bind tubes and dilute 1:1 with 100 &#181;L of 30% (v/v) glycerol in 1x PBS. Flash freeze with liquid nitrogen and store at -80 &#176;C. These samples will be the Day 0 timepoint, and the concentration of HsADA1 in them will be 500 nM. Repeat this step for the non-enzyme control mixtures from 1.3. Seal all enzymes and control mixtures with transparent film and place them into a 37 &#176;C incubator.</li>
	<li>After 24 h, aliquot 100 &#181;L of each incubated sample and dilute 1:1 with 100 &#181;L of 30% (v/v) glycerol in 1x PBS. Flash freeze with liquid nitrogen and store at -80 &#176;C. These samples will be the Day 1 timepoint. Return all enzyme and control mixtures to the 37 &#176;C incubator. Repeat this step at 72 h and 120 h. 
	NOTE: Aliquoted samples do not necessarily need to be flash-frozen. Alternatively, the user may perform the microplate-based assay immediately after taking each sample, although fresh standard curves and adenosine substrate stocks should be prepared each time a sample is tested.</li>
</ol>
2. Microplate-based assay
<ol>
	<li>Prepare the microplate reader by setting up a kinetic read protocol (Figure 2) to measure the absorbance at 260 nm with the shortest reading interval for 30 min and preheat the reader to a setpoint of 37 &#176;C.</li>
	<li>Thaw all collected samples on ice. Dilute each thawed sample 1:10 using 1x PBS prewarmed to 37 &#176;C as a diluent in low-bind microcentrifuge tubes. The new HsADA1 concentration in these samples will be 50 nM.</li>
	<li>Make a 10x dilution into 1x PBS from the non-enzyme serum control sample as well. Additionally, prepare a 5 mM adenosine stock by adding 66.8 mg of adenosine to 50 mL of 1x PBS. Prepare a 250 &#181;M dilution of adenosine by adding 400 &#181;L of the adenosine stock to 7,600 &#181;L of 1x PBS and prewarm it to 37 &#176;C in an incubator.</li>
	<li>To a 96-well UV-compatible microplate, add 160 &#181;L of the 250 &#181;M adenosine dilution and 40 &#181;L of each diluted protein sample in triplicate for a total volume of 200 &#181;L. This will yield a final adenosine concentration of 200 &#181;M and a final enzyme concentration of 10 nM.</li>
	<li>Repeat the dilution for the control samples by adding 40 &#181;L of the diluted serum or 1x PBS controls and 160 &#181;L of the 250 &#181;M adenosine dilution in triplicate. This will also yield a final adenosine concentration of 200 &#181;M but without enzyme. 
	NOTE: It may be beneficial to add all diluted enzyme and control samples individually to the microplate first and then add the adenosine substrate with a 12-channel micropipette to reduce the total loading time. Additionally, UV-transparent plates must be used in this step to prevent the plate itself from confounding the measured absorbance signal.</li>
	<li>Measure the absorbance of each well at 260 nm for 30 min at 37 &#176;C. Once done, export the data to a spreadsheet for further analysis.</li>
</ol>
3. Analysis of microplate reader data
<ol>
	<li>Determine the slope of the linear region of the absorbance data as a function of time for the first several minutes of the microplate data. For the serum-diluted samples, this corresponded to the first ~120 s. For the 1x PBS-diluted samples, this corresponded to the first ~270 s. This slope of declining absorbance values will have a negative numerical value and will have units of change in absorbance units per second (&#916;A260/s).</li>
	<li>Subtract out the slope of the negative control consisting of pooled human serum from the slope of the enzyme + serum data. Perform the same operation on the enzyme + 1x PBS mixtures by subtracting out the slope of the 1x PBS control.</li>
	<li>Normalize all the adjusted slopes to the original slope at t=0 h to obtain the fraction of activity remaining using Equation 1. 
	<img alt="Equation 1" src="/files/ftp_upload/67216/67216eq01.jpg" style="margin: auto;" />&#160; &#160; Equation 1</li>
	<li>Plot the fraction of activity remaining versus time.</li>
</ol>

Evaluating Serum Stability of HsADA1: A Microplate-Based Approach

<ol>
	<li>Valero, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+serum-stable+RNA+aptamer+specific+for+SARS-COV-2+neutralizes+viral+entry.">A serum-stable RNA aptamer specific for SARS-COV-2 neutralizes viral entry.</a> Proc Natl Acad Sci U S A. 118 (50), e2112942118 (2021).</li><li>Iannuzo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+identifies+synthetic+peptides+with+antibacterial+activity+against+mycobacterium+abscessus+and+serum+stability.">High-throughput screening identifies synthetic peptides with antibacterial activity against mycobacterium abscessus and serum stability.</a> ACS Omega. 7 (27), 23967-23977 (2022).</li><li>Ma, M. T., Jennings, M. R., Blazeck, J., Lieberman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytically+active+holo+homo+sapiens+adenosine+deaminase+i+adopts+a+closed+conformation.">Catalytically active holo homo sapiens adenosine deaminase i adopts a closed conformation.</a> Acta Crystallograph Sect D. 78 (1), 91-103 (2022).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+4+nucleosides+via+one+reference+compound+in+chinese+cordyceps+by+hplc-uv+at+equal+absorption+wavelength.">Determination of 4 nucleosides via one reference compound in chinese cordyceps by hplc-uv at equal absorption wavelength.</a> Natural Prod Comm. 18 (3), 1934578X231161410 (2023).</li><li>Whitmore, K. V., Gaspar, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+deaminase+deficiency+-+more+than+just+an+immunodeficiency.">Adenosine deaminase deficiency - more than just an immunodeficiency.</a> Front Immunol. 7, 314 (2016).</li><li>Jennings, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+expression+and+purification+of+a+human+adenosine+deaminase+in+E.+coli+and+characterization+of+its+asp8asn+variant.">Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its asp8asn variant.</a> Prot Express Purificat. 213, 106362 (2024).</li><li>Blazeck, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bypassing+evolutionary+dead+ends+and+switching+the+rate-limiting+step+of+a+human+immunotherapeutic+enzyme.">Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.</a> Nat Catalysis. 5 (10), 952-967 (2022).</li><li>Lu, J., Grenache, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rapid,+microplate-based+kinetic+assay+for+measuring+adenosine+deaminase+activity+in+body+fluids.">Development of a rapid, microplate-based kinetic assay for measuring adenosine deaminase activity in body fluids.</a> Clinica Chimica Acta. 413 (19), 1637-1640 (2012).</li><li>Maciel, L. G., Dos Anjos, J. V., Soares, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+low-cost+evaluation+of+hydroxykynurenine+activity.">Fast and low-cost evaluation of hydroxykynurenine activity.</a> MethodsX. 7, 100982 (2020).</li><li>Bokman, A. H., Schweigert, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-hydroxyanthranilic+acid+metabolism.+Iv.+Spectrophotometric+evidence+for+the+formation+of+an+intermediate.">3-hydroxyanthranilic acid metabolism. Iv. Spectrophotometric evidence for the formation of an intermediate.</a> Arch Biochem Biophys. 33 (2), 270-276 (1951).</li><li>Tanford, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in protein chemistry. 23, 121-282 (1968).</li><li>Tritsch, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validity+of+the+continuous+spectrophotometric+assay+of+kalckar+for+adenosine+deaminase+activity.">Validity of the continuous spectrophotometric assay of kalckar for adenosine deaminase activity.</a> Anal Biochem. 129 (1), 207-209 (1983).</li><li>Gracia, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+catalytic+site+structural+gate+of+adenosine+deaminase+allosterically+modulates+ligand+binding+to+adenosine+receptors.">The catalytic site structural gate of adenosine deaminase allosterically modulates ligand binding to adenosine receptors.</a> FASEB J. 27 (3), 1048-1061 (2013).</li><li>Kalckar, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+spectrophotometry+of+purine+compounds+by+means+of+specific+enzymes:+Iii.+Studies+of+the+enzymes+of+purine+metabolism.">Differential spectrophotometry of purine compounds by means of specific enzymes: Iii. Studies of the enzymes of purine metabolism.</a> J Bio Chem. 167 (2), 461-475 (1947).</li><li>Hartwick, R., Jeffries, A., Krstulovic, A., Brown, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+assay+for+adenosine+deaminase+using+reverse+phase+high+pressure+liquid+chromatography.">An optimized assay for adenosine deaminase using reverse phase high pressure liquid chromatography.</a> J Chromatographic Sci. 16 (9), 427-435 (1978).</li><li>Paul, M. K., Grover, V., Mukhopadhyay, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merits+of+hplc-based+method+over+spectrophotometric+method+for+assessing+the+kinetics+and+inhibition+of+mammalian+adenosine+deaminase.">Merits of hplc-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase.</a> J Chromatography B. 822 (1), 146-153 (2005).</li><li>Ubbink, J. B., Vermaak, W. J. H., Bissbort, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+liquid+chromatographic+assay+of+human+lymphocyte+kynureninase+activity+levels.">High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels.</a> J Chromatography B: Biomed Sci Appl. 566 (2), 369-375 (1991).</li><li>Tsentalovich, Y. P., Snytnikova, O. A., Forbes, M. D. E., Chernyak, E. I., Morozov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochemical+and+thermal+reactivity+of+kynurenine.">Photochemical and thermal reactivity of kynurenine.</a> Exp Eye Res. 83 (6), 1439-1445 (2006).</li><li>Demain, A. L., Vaishnav, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+by+microbes+and+higher+organisms.">Production of recombinant proteins by microbes and higher organisms.</a> Biotechnol Adv. 27 (3), 297-306 (2009).</li><li>Gr&#228;slund, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+and+purification.">Protein production and purification.</a> Nat Methods. 5 (2), 135-146 (2008).</li><li>Faber, M. S., Whitehead, T. 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JB and MRJ are inventors on patents or patent applications related to adenosine deaminase and/or kynureninase enzymes. All other authors have no conflicts of interest to disclose.

This protocol uses absorbance change as the substrate is converted to the product to gauge the activity of an enzyme. As such, the substrate and product must have distinct spectral profiles. This is the case with adenosine and inosine both having distinct spectral profiles and extinction coefficients between 260-265 nm6,8,12,13. This assay is inspired by several previous works. Kalackar, for ex...

The following method allows a user to quantitatively assess an enzyme&#39;s ability to retain its activity when exposed to conditions that mimic what it will encounter following intravenous injection. The in vitro method mimics such in vivo conditions and consists of the incubation of the enzyme in pooled human serum at 37 &#176;C and time-coursed analyses of retention of enzyme activity. We refer to an enzyme&#39;s ability to retain activity in these conditions as its serum stability, and the analysis method for enzyme activity takes advantage of differences in absorbance between an enzyme&#39;s substrate and the resulting product. The concept of serum stability is not just enzyme-specific and has been applied to several other treatment modalities as well. For example, the serum stability of RNA aptamers targeting COVID spike proteins has previously been assessed by monitoring their degradation post-incubation with fetal calf serum1. Antibacterial peptides have also been assessed for their ability to suppress bacterial growth post-incubation with pooled human serum2.
HsADA1 is an enzyme that catalyzes the conversion of adenosine or 2-deoxyadenosine into inosine or 2-deoxyinosine, respectively3. Adenosine has an absorption peak of 260 nm, while inosine absorbs strongest at 250 nm4. This shift in absorption peaks can be detected on a microplate reader by a decrease in absorption intensity at 260 nm when HsADA1 is added to adenosine. The HsADA1 enzyme has important implications in the human body, and its deficiency causes a severe combined immunodeficiency (ADA-SCID)5. The bovine homolog of HsADA1, BtADA1, can be utilized as an enzyme replacement therapeutic for the treatment of ADA-SCID, and we have previously shown that wildtype HsADA1 loses its activity when incubated with pooled human serum, potentially hindering its use as a therapeutic6. Therefore, we selected the wildtype HsADA1 enzyme to demonstrate a procedure to determine an enzyme&#39;s serum stability. A detailed purification method for HsADA1 has been described previously6.
In the following protocol (as detailed in Figure 1), we demonstrate how to co-incubate wildtype HsADA1 in pooled human serum at 37&#160;&#176;C. During this time, test samples are taken at defined time points and flash-frozen for future analysis. Once all samples have been taken, a microplate assay is run whereby each sample is combined with the substrate, with the resulting absorbance decrease being a correlation for the retained activity of the enzyme. Representative results illustrating the serum stability of HsADA1 are shown, and because this metric may be relevant to determining the potential therapeutic value of other enzymes, we also discuss considerations to adapt this protocol to an engineered Homo sapiens kynureninase enzyme (HsKYNase) and any enzymes more generally. HsKYNase is an enzyme involved in the metabolism of tryptophan and is able to degrade the tryptophan-byproducts kynurenine and hydroxy-kynurenine (OH-Kyn) into anthranilic acid and hydroxy-anthranilic acid (OH-AA), respectively. Enzyme-mediated modulation of tryptophan metabolism may be of therapeutic relevance7.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>A9251-25G</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>BioTek Synergy HT Microplate Reader</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf LoBind Microcentrifuge Tubes: Protein&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-698-795</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific&#160;</td>
			<td>G33-4</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>HsKYNase66-W102H-T333N</td>
			<td>In-house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum, Pooled</td>
			<td>MP Biomedicals</td>
			<td>92931149</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Hydroxy-kynurenine</td>
			<td>Cayman Chemicals</td>
			<td>27778</td>
			<td></td>
		</tr>
		<tr>
			<td>Inosine&#160;</td>
			<td>TCI</td>
			<td>I0037</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>PBS, 1x&#160; pH 7.4+/- 0.1</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal 5-phosphate monohydrate, 99%&#160;</td>
			<td>Thermo Scientifc</td>
			<td>228170010</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>UV-STAR MICROPLATE, 96 WELL, COC, F-BOTTOM</td>
			<td>Greiner Bio</td>
			<td>&#160;655801</td>
			<td></td>
		</tr>
		<tr>
			<td>Wildtype Human Adenosine Deaminase 1</td>
			<td>In-house</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

determining the serum stability of human adenosine deaminase 1 enzyme

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature increases. For a therapeutic enzyme, other measures of stability may also be critical, particularly its ability to retain function in human serum at 37 &#176;C, which we refer to as serum stability. Here, we describe an in vitro assay to assess the serum stability of the wildtype Homo sapiens adenosine deaminase I (HsADA1) enzyme using an absorbance-based microplate procedure. Specifically, this manuscript describes the preparation of buffers and reagents, a method arranging for the coincubation of HsADA1 in serum, a method to analyze the test samples using a microplate reader, and an accompanying analysis to determine the fraction of activity that an HsADA1 enzyme retains in serum as a function of time. We further discuss considerations to adapt this protocol to other enzymes, using an example of a Homo sapiens kynureninase enzyme, to help aid the protocol's adaptation to other enzymes where serum stability is of interest.

The figures show the results of the assay run when conducted with wildtype HsADA1. Figure 3A,B illustrate the absorbance decline curves at 260 nm of the samples originating from the 1x PBS/serum-enzyme mixtures for wildtype HsADA1 after the addition of adenosine. This declining absorbance as a function of time data is what the user may expect upon successful completion of the microplate-based assay and is similar to absorbance data that would arise after adding sufficient am...

Author Spotlight: Advancing Cellular and Protein Engineering to Control Biological Functions and Develop Novel Therapies

In this article, we detail methods to characterize an enzyme&#39;s ability to retain function when incubated at 37&#160;&#176;C in human serum, a pharmacological property referred to as its serum stability. This ability may be a key factor in predicting an enzyme&#39;s pharmacokinetic profile and its suitability for therapeutic use.

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature ...

So our lab works at the interface of immunology, engineering, and metabolism to improve human health. We use our expertise in protein and cellular engineering to make new therapies and control biological functions. If researchers desire to increase the circulating half-life of protein biologics, this protocol could help inform users whether serum stability is a rate limiting step.Understanding this key point would help guide future protein engineering efforts to the area of need. One question we'll explore is whether proteins with enhanced serum stability result in greater therapeutic efficacy in the context of treating cancer than their non engineered counterparts. We'll further explore whether therapeutic efficacy can be achieved using less doses due to this enhanced stability.

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477 Views.  Georgia Institute of Technology. So our lab works at the interface of immunology, engineering, and metabolism to improve human health. We use our expertise in protein and cellular engineering to make new therapies and control biological functions. If researchers desire to increase the circulating half-life of protein biologics, this protocol could help inform users whether serum stability is a rate limiting step.Understanding this key point would help guide future protein engineering efforts to the area of need. One q...

Video: Author Spotlight: Advancing Cellular and Protein Engineering to Control Biological Functions and Develop Novel Therapies

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...