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Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen Roberta Polak, Grinia Michelle Bradwell, Jonathan Brian Gilbert, Scott Danielsen, Marisa Masumi Beppu, Robert E. Cohen, and Michael F. Rubner Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00443 • Publication Date (Web): 24 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen Roberta Polak†¥, Grinia M. Bradwell¥, Jonathan B. Gilbert§, Scott DanielsenϮ, Marisa M. Beppu‡, Robert E. Cohen§, Michael F. Rubner¥*. † School of Pharmaceutical Sciences, University of Sao Paulo, USP, Sao Paulo/SP, Brazil ‡ School of Chemical Engineering, University of Campinas, UNICAMP, Campinas/SP, Brazil Ϯ Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia/PA, USA § Department of Chemical Engineering, Massachusetts Institute of Technology, MIT, Cambridge, USA ¥ Department of Materials Science and Engineering, Massachusetts Institute of Technology, MIT, Cambridge, USA
KEYWORDS: layer-by-layer, polymer functionalization, biotinylation, prostate-specific antigen, swelling. 1 ACS Paragon Plus Environment
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ABSTRACT. In this work, it is demonstrated that poly(allylamine hydrochloride)/poly(styrene sulfonate) (PAH/SPS) multilayer films can be successfully tailored for the capture and detection of small biomolecules in dilute concentrations. Based on in vitro results, these films could be potentially applied for rapid and high-throughput diagnosis of dilute biomarkers in serum or tissue. PAH presents functional amino groups that can be further reacted with desired chemistries in order to create customizable and specific surfaces for biomolecule capture. A variety of film assembly characteristics were tested (pH, molecular weight of PAH, and ionic strength) to tune the biotinylation and swelling behavior of these films to maximize detection capabilities. The resultant optimized biotinylated PAH/SPS 9.3/9.3 system was utilized in conjunction with quantum dots (Qdots) to capture and detect a dilute biomarker for prostate cancer, prostatespecific antigen (PSA). Compared to previous work, our system presents a good sensitivity for PSA detection within the clinically relevant range of 0.4 ng/mL to 100 ng/mL.
Introduction Polyelectrolyte multilayer (PEM) films are capable of delivering conformal coatings on geometrically confined or complex spaces.1-3 Such coatings, for example, have been developed for substrates ranging from standard solid surfaces to highly porous membranes,4-5 degradable scaffolds,6-7 microneedles,8-9 carbon nanotubes,10-12 and microfluidic devices.13-15 By depositing PEM films onto these substrates, one can generate unique functionalities by taking advantage of the chemical diversity and flexibility inherent in PEM films. Simply by modifying the film with chemistries of interest16 it is possible to generate functional films for a wide range of applications 2 ACS Paragon Plus Environment
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including the use of functional membranes in nanofiltration or gas-separation,17 functional membranes for protein adsorption or catalysis,18-19 endothelialization,20 sensors,21 and especially conjugation with antibodies.22 Such biofunctional coatings have already seen applications in promoting endothelialization and cell growth,23-24 the capture and detection of circulating tumor cells,25 and protein-based nanotubes for the selective capture of nanoparticles, proteins and viruses.22 The detection of dilute protein biomarkers can facilitate early disease diagnosis and consequently treatment, thereby improving patient survival.26 An example of this potential is with prostate specific antigen (PSA). PSA is a blood biomarker secreted by normal and tumor cells27-28 with a concentration of ~ 4 ng/mL in serum28 having been attributed to the possible presence of prostate cancer. However, the detection of PSA alone is not decisive for a cancer diagnosis,29 nor can the PSA concentration be correlated with tumor size.30 Although the detection of PSA in serum must be combined with examination and a prostate biopsy, the detection of low concentrations of PSA remains widely studied for cancer detection and may be an effective tool for early diagnosis. Lab miniaturization could replace conventional protein detection techniques with more cost effective and higher throughput techniques. This has led to increased efforts to fabricate such point-of-care diagnosis devices.26, immunosorbent
assays,
or
31
other
Although gold standard techniques like enzyme-linked viable
approaches
such
as
those
based
on
electrochemiluminescence, electrophoresis, or proteomics have acceptable sensitivities, they typically require user expertise and may be prohibitively costly for widespread use. One promising example is the electrochemical detection of proteins, which usually uses an EDC-NHS amidification to anchor specific antibodies to a material surface.32-33 Such devices have been 3 ACS Paragon Plus Environment
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recently investigated in the capture of important biomarkers like interleukin-6 and prostate specific antigen.33-34 Herein, we studied the parameters that govern the availability of amine functional groups in PEM films for functionalization and PSA detection. Although there are numerous examples of PEM films functionalized post-assembly via the use of free carboxylic acid groups,35-42 the creation and functionalization of reactable amine groups is much less explored due to the difficulty of producing multilayer films with free, non-polymer anion bound ammonium groups.38 Thus, we explored and optimized parameters for maximizing amine functionalization in a well studied multilayer film architecture of poly(allylamine hydrochloride)/poly(styrene sulfonate), PAH/SPS.43-50 We applied optimal film parameters for the creation of biotinylated multilayers for the capture and detection of PSA protein, by using Qdots as probes.51 The resultant system presents a good sensitivity for PSA detection that is within the range of 0.4 ng/mL to 100 ng/mL.27, 52-53
Experimental Section Material: PAH: Poly(allylamine hydrochloride), 56,000 g/mol or 15,000 g/mol (Aldrich). SPS: Poly(styrene sulfonate), 70,000 g/mol (Sigma-Aldrich). Fluor: Fluorescein sodium salt (Sigma), NHS-Fluor: N-hydroxysuccinimide ester activated fluorescein (Pierce Biotechnology); NHSBiotin: (+)-Biotin N-hydroxysuccinimide ester (Sigma Aldrich); STA: Streptavidin (Pierce Biotechnology); Neutravidin protein (Pierce Biotechnology); Qdot® 655 Streptavidin Conjugate (Life Technologies); BSA: Albumin from bovine serum (Sigma Aldrich); PSAab: Monoclonal Antibody against Prostate-specific antigen, Biotin conjugate (Pierce Biotechnology); PSAag:
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Prostate Specific Antigen, human (Millipore); PBS: Phosphate-Buffered Saline (Corning Cellgro); FBS: fetal bovine serum (Life Technologies). LbL assembly: Polymer solution concentrations were 10 mM (based on repeat unit) with either 0 or 0.1 M NaCl. Polymers and deionized water rinse solutions were adjusted to desired pH using 1 M HCl or 1 M NaOH solutions. The PAH/SPS system was assembled at either pH 3.0 or 9.3 using a spin-assisted dipper StratoSequence VI, NanoStrata Inc. Glass slides or Si wafer were used as substrates and dipped into polymer solutions until the desired number of bilayers was reached (15.5 or 5.5 bilayers). The substrate was dipped for 10 minutes for the polymer solution followed by three rinse steps of 2, 2 and 1 minute each. Rinse water was adjusted to the same pH of the polymer solutions and no salt was added (except for the experiments using salt in all steps, where 0.1 M NaCl was added). One bilayer is comprised of one polycation (PAH) and one polyanion (SPS) layer. The polycation was assembled as the first and last layer, to increase the amine groups on the film surface. Films were used as deposited (AD) or after performing a postassembly acid treatment (AT) exposure to pH 2.5 for 15 min. Samples were rinsed twice in deionized water to remove any acid traces and dried with compressed air. All films used in this work were used within 10 hours after assembly. Determination of thicknesses in dry and wet state: Thicknesses and swelling degree of the PEMs films were measured using a spectroscopic ellipsometer (Woollam M-2000D; ISN, MIT). All thickness measurements were made at three different points on each film and averaged. For the swelling measurements, films were placed inside a fluid chamber.54 Note that in this case, the swelling measurements were taken in deionized water at pH 7 and in PBS for as-deposited or for pH treated (pH 2.5 or pH 10), rinsed and dried films. First, the pH treated rinsed and dried film thicknesses were measured. Then, the desired solution (deionized water or PBS) was added into 5 ACS Paragon Plus Environment
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the fluid chamber. After 5 minutes the swollen measurement was taken (we observed that after 5 minutes the swelling stabilized for all films). The percentage of swelling of the multilayers films can be defined as: % Swelling = 100(Tin solution – Tdry) / Tdry
(1)
Where Tdry is the thickness of the dried film and Tin solution is the film thickness after swelling. Post-assembly NHS functionalization: NHS-esters (NHS-Biotin and NHS-Fluor) were diluted into aliquots in DMSO (stock solution 20 mM) and stored at -20 °C. Prior to analyses the aliquots were thawed at room temperature and further diluted to 5 mM in PBS buffer. LbL films were reacted for 2 hours at room temperature, and then rinsed twice in PBS buffer and air-dried. To assess the reaction efficiency, we reacted NHS-Fluorescein (NHS-Fluor; 5 mM in PBS) to the different surfaces using Fluorescein sodium salt (Fluor; 5 mM in PBS) solution as a control for non-specific adsorption. After the reaction, we looked at the absorbance spectrum of the samples via UV-Vis spectroscopy. UV-vis spectra were acquired in the range of 450 to 600 nm (Varian Cary 5E spectrophotometer); an average of 4 samples were used. All solutions were adjusted to desired pH using 1 M NaOH solution before reaction. The absorbance data at 540 nm was averaged for each sample. For statistical analyses, t-tests with 95% confidence interval were performed using Prism 5 software. X-ray Photoelectron Spectroscopy (XPS): XPS was performed to determine the amount of amine and ammonium groups in the films. Chemical composition of approximately the top 10nm of the film was characterized using a PHI VersaProbe II X-ray photoelectron spectrometer with a scanning monochromated Al source (1,486.6 eV; 50 W; spot size, 200 μm). The takeoff angle between the sample surface and analyzer was 45°, and the X-ray beam collected C1s, N1s, O1s,
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and Si2p elemental information while rastering over a 200Χ700-μm area. Peak fitting analysis was done in CasaXPS. Qdots capture by biotinylated films: In order to simulate the capture of small biological particles (e.g. proteins), a validation experiment was performed using streptavidin coated Qdots. Films reacted with NHS-Biotin (pre or post assembled) were incubated with 20 nM of Qdots diluted in blocking buffer (PBS containing 6% of BSA). For control samples as deposited (AD) and acid treated (AT) reacted films were used. All samples were incubated with blocking buffer for 1h followed by two rinse steps in PBS, before reaction with Qdots. The use of blocking buffer was crucial to avoid the adsorption of Qdots by non-specific interactions on nonbiotinylated films. Streptavidin conjugated Qdots (STA-Qdots) were reacted for 2h at RT and rinsed twice in PBS buffer. Samples were observed in a fluorescence microscope, and the fluorescence intensity was quantified with Image J for each sample. An average of 3 experiments was used. PSA capture: Biotinylated films were incubated with blocking buffer for 1h, rinsed three times in PBS and then reacted with Neutravidin (50 µg/mL, 1 h, 4 ºC). Of note, we have examined many different conditions until reaching the best parameters of the current capture protocol. We found that using the blocking buffer prior to incubation of both neutravidin and PSA antigen helped prevent non-specific adsorption of the subsequent reagents. Three rinse steps in PBS were performed to remove unreacted Neutravidin. A solution of PSA biotinylated antibody (4 µg/mL) was reacted for 30 min, followed by three rinse steps in PBS. Another step of blocking buffer was performed previous to PSA capture in order to prevent nonspecific interactions. PSA antigen at desired concentration (100 ng/mL to 0.1 ng/mL) was diluted in either PBS buffer or fetal bovine serum (FBS, passed through 0.2 µm filters prior dilution of PSA), and then incubated for 7 ACS Paragon Plus Environment
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2 minutes followed by three rinse steps in PBS. PSA biotinylated antibody was reacted to the PSA antigen for 2 minutes followed by three rinse steps in PBS. Finally, 20 nM of streptavidin conjugated Qdots were incubated for 5 minutes to react with the biotinylated antibody. Samples were rinsed thrice in PBS and examined in a fluorescence microscope. The fluorescence intensity was quantified with Image J software. An average of 3 experiments was used. SEM was performed on 100 ng/mL of reacted antigen for biotinylated acid treated samples and on nonbiotinylated samples (control). Samples were coated with 5 ng of carbon before imaging.
Results and discussion Effects of molecular weight, ionic strength and film post treatment on film functionalization For the detection of the biomarker PSA at dilute concentrations in solution, it is important to maximize the ability for its capture. One way to achieve this is to substantially increase the density of PSA antibodies present within the PEM film. Thus, the first step in our investigation was to study the parameters related to reactive amine availability in a PEM film system with regards to functionalization for the capture of dilute antigens in solution. Using a PAH/SPS 9.3/9.3 system, the polycation poly(allylamine hydrochloride) (PAH, Figure 1) presents free primary amine groups that can be modified through simple amide-forming chemistry with a variety of reagents. PAH is a weak polycation with a pKa ~ 8.855 and by tuning the pH of assembly one can adjust its charge density, leading to fine control over the thickness of each deposited layer.56-57 As previously demonstrated by our group,44 PAH assembled with the strong polyelectrolyte poly(styrene sulfonate) (SPS, Figure 1) at pH 9.3 combines a partially ionized polycation with a fully ionized polyanion generating a film in which the non-ionized portions of PAH preferentially organize into hydrophobic pockets. When undergoing acid treatment (AT) by 8 ACS Paragon Plus Environment
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incubation in solutions of pH ≤ 4 for 15 minutes, films of the PAH/SPS 9.3/9.3 system demonstrate a high swelling capability that is not observed for the as deposited (AD) films. Acid treatment of the 9.3/9.3 films protonates the primary amines of PAH trapped in the hydrophobic pockets and causes their rearrangement within the film for greater solvent accessibility and consequently increased hydrophilicity that promotes additional swelling capability.44
(PAH)
(SPS)
Figure 1. Structural formulas of poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (SPS). In order to characterize the swelling capacity, the state of amine ionization and its influence on functionalizability, we evaluated a number of assembly parameters in the PAH/SPS 9.3/9.3 system and their effects on amine derivatization using N-hydroxysuccinimidyl ester (NHS) conjugation chemistry. Three different parameters were evaluated: the influence of ionic strength of solutions during assembly (no salt or 0.1 M NaCl), the molecular weight of PAH (15 or 56 kDa), and the effect of a post-assembly acid treatment. Growth curves (Figure S1, Table S1) for all multilayers tested were linear, giving a good control over the film thickness depending on the number of deposited bilayers. A detailed discussion regarding the parameters and their influence on the relative degrees of functionalization can be found in Supporting Information (Figure S2). Acid treated multilayer films were reacted with an NHS-ester of fluorescein, NHS-Fluor, and the resultant fluorescein
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absorbance was used as an indicator of the relative accessibilities of the primary amines. A summary of results can be found in Figure 2.
% of Increase in Fluorescein conjugation after acid treatment
40
PAH 15 kDa PAH 56 kDa 30
20
10
0
No salt
0.1 M NaCl in polymer solution only
0.1 M NaCl in polymer and rinse solutions
Sa lte dP oly /Ri ns e
Sa lte dP oly
Figure 2. Effect of PAH molecular weight and salt in rinse and/or polymer solutions on NHSNo Sa lt
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functionalization (performed at pH 8.3 for 2h) as measured by fluorescein conjugation after different assembly conditions. Data acquired by UV-Vis absorbance at 540 nm.
Fluorescein conjugation increased after acid treatment (AT) for all assembly ionic strength parameters (p < 0.05) as compared to as-deposited films (AD) films. This enhancement of fluorescein conjugation corresponds with the increased accessibility of free amines that is generated after acid treatment.44 Most notably, acid treatment of multilayer films assembled with salt and especially salt in the polymer solutions only resulted in significantly greater amounts of amine fluorescein functionalization, as observed in Figure 2 (See detailed discussion in Supporting Information, Figure S2). Previous studies by us with this multilayer system did not include salt in the polymer solutions. The addition of salt in the polymer assembly solutions clearly improves the functionalization capacity. In addition, by increasing the molecular weight of PAH from 15 kDa to 56 kDa, amine functionalization is improved (Figure S2). Previous 10 ACS Paragon Plus Environment
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works have shown that higher molecular weight polymers tend to arrange with a larger fraction of segmental loops when compared to shorter polymers, and therefore promote larger thickness increments per deposited bilayer.58-59 In this case, we hypothesize that the more loopy molecular organization created with higher molecular weight PAH facilitates more amine enriched hydrophobic pockets compared to the lower molecular weight PAH. To further investigate the parameters surrounding conjugation of the available amines, we varied the pH during fluorescein conjugation of films assembled with the optimal conditions identified above: high molecular weight PAH (56kDa), and salt in polymer solutions (no salt in rinse solutions). 0.20
pH 8.3 Reaction
pH 9 Reaction
pH 10 Reaction
Absorbance (a.u.)
0.18 0.16
*
0.14 0.12 0.10 0.08 0.06 0.04
A AD D N Flu H S- or Fl u A AT or T F N l H uo S- r Fl u A AD or D F N l H uo Sr Fl u A AT or T N Flu H S- or Fl u A AD or D F N l H uo Sr Fl u o r A AT T N Flu H S- or Fl uo r
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Figure 3. Effect of pH of conjugation reaction on PAH/SPS 9.3/9.3 15.5 bilayer films (PAH: 56 kDa / SPS: 70 kDa). All films assembled with 0.1 M NaCl in polymer solutions. As controls to determine non-specific interactions, films were treated under the same conditions with fluorescein sodium salt (Fluor) at 5mM for 2 h at room temperature. Absorbance at 540 nm was acquired by UV-vis. AD: as deposited films; AT: acid treated films. 11 ACS Paragon Plus Environment
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As observed in Figure 3, when fluorescein conjugation is performed with solution pHs in the range of 8 to 10, there is a greater degree of functionalization of the acid treated films than that of as-assembled films and the control samples: with pH 10 providing the highest level of functionalization. This improvement in functionalization suggests that there is a larger fraction of free amines present in their more reactive, non-ionized (and hence more nucleophilic) form. As noted above, this functionalization was significantly greater than that obtained by an NHSFluor reaction of as-assembled films, as well as the AD and AT films incubated with Fluor, which was used as a control for determining non-specific interactions.
Surface Composition of PAH/SPS films on different pH environments To further evaluate the surface composition of PAH/SPS films, we used X-ray Photoelectron Spectroscopy (XPS) to determine the ratio of amine and ammonium groups resulting from differences in pH of film deposition (pH 3.0 or 9.3) and post-assembly pH treatments. This technique allows for analysis of the elemental sample composition in the realm of 0-10 nm beneath the surface. Analysis was performed on samples fabricated with high molecular weight PAH (56kDa), and salt in polymer solutions (no salt in rinse solutions).
Table 1. Ratio of amine/ammonium groups available on film surfaces. Data obtained by XPS analyses (See Figure S3 for fitting example).
Sample (PAH3.0/SPS3.0)15.5
Amine (%)
Ammonium (%)
21
79 12
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(PAH9.3/SPS9.3)15.5 as deposited
44
56
(PAH9.3/SPS9.3)15.5 acid treated (pH 2.5, 15 min)
20
80
(PAH9.3/SPS9.3)15.5 acid treated + incubation at pH 8.3 for 2h
21
79
(PAH9.3/SPS9.3)15.5 acid treated + incubation at pH 9 for 2h
44
56
(PAH9.3/SPS9.3)15.5 acid treated + incubation at pH 10 for 2h
46
54
As detailed in Table 1, the as deposited 9.3/9.3 films contain about 56% of nitrogen groups present as ammonium groups. Subsequent acid treatment induces an increase in the ammonium fraction to 80%, as contact with pH 2.5 shifts the equilibrium towards protonation of the free nitrogen groups, including those contained in hydrophobic clusters. Taking the acid treated film and incubating it for 2h in pH 8.3 does not substantially affect the fraction of ammonium groups (79%), but incubation in pH 9 or 10 causes a significant shift of nitrogen groups into non-ionized amines. In previous infrared spectroscopy examinations of PAH9.3/SPS9.3 multilayer films assembled without salt,43 we observed slightly greater degrees of ionization for the nitrogen groups of PAH in as deposited films (70%),43 and acid treated films (90%). These slight differences may be due to salt shielding resulting in a smaller fraction of the total nitrogen groups of PAH to participate in electrostatic crosslinks with SPS within the film. The tendency for nitrogen groups participating in such electrostatic crosslinks to remain ionized beyond pH conditions that would deprotonate otherwise free nitrogen groups, could reflect the greater presence of ammonium groups in the previous salt-free films.60 Also, one should note that this difference might be observed as XPS analyzes the near-surface (
Article
Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen Roberta Polak, Grinia Michelle Bradwell, Jonathan Brian Gilbert, Scott Danielsen, Marisa Masumi Beppu, Robert E. Cohen, and Michael F. Rubner Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00443 • Publication Date (Web): 24 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen Roberta Polak†¥, Grinia M. Bradwell¥, Jonathan B. Gilbert§, Scott DanielsenϮ, Marisa M. Beppu‡, Robert E. Cohen§, Michael F. Rubner¥*. † School of Pharmaceutical Sciences, University of Sao Paulo, USP, Sao Paulo/SP, Brazil ‡ School of Chemical Engineering, University of Campinas, UNICAMP, Campinas/SP, Brazil Ϯ Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia/PA, USA § Department of Chemical Engineering, Massachusetts Institute of Technology, MIT, Cambridge, USA ¥ Department of Materials Science and Engineering, Massachusetts Institute of Technology, MIT, Cambridge, USA
KEYWORDS: layer-by-layer, polymer functionalization, biotinylation, prostate-specific antigen, swelling. 1 ACS Paragon Plus Environment
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ABSTRACT. In this work, it is demonstrated that poly(allylamine hydrochloride)/poly(styrene sulfonate) (PAH/SPS) multilayer films can be successfully tailored for the capture and detection of small biomolecules in dilute concentrations. Based on in vitro results, these films could be potentially applied for rapid and high-throughput diagnosis of dilute biomarkers in serum or tissue. PAH presents functional amino groups that can be further reacted with desired chemistries in order to create customizable and specific surfaces for biomolecule capture. A variety of film assembly characteristics were tested (pH, molecular weight of PAH, and ionic strength) to tune the biotinylation and swelling behavior of these films to maximize detection capabilities. The resultant optimized biotinylated PAH/SPS 9.3/9.3 system was utilized in conjunction with quantum dots (Qdots) to capture and detect a dilute biomarker for prostate cancer, prostatespecific antigen (PSA). Compared to previous work, our system presents a good sensitivity for PSA detection within the clinically relevant range of 0.4 ng/mL to 100 ng/mL.
Introduction Polyelectrolyte multilayer (PEM) films are capable of delivering conformal coatings on geometrically confined or complex spaces.1-3 Such coatings, for example, have been developed for substrates ranging from standard solid surfaces to highly porous membranes,4-5 degradable scaffolds,6-7 microneedles,8-9 carbon nanotubes,10-12 and microfluidic devices.13-15 By depositing PEM films onto these substrates, one can generate unique functionalities by taking advantage of the chemical diversity and flexibility inherent in PEM films. Simply by modifying the film with chemistries of interest16 it is possible to generate functional films for a wide range of applications 2 ACS Paragon Plus Environment
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including the use of functional membranes in nanofiltration or gas-separation,17 functional membranes for protein adsorption or catalysis,18-19 endothelialization,20 sensors,21 and especially conjugation with antibodies.22 Such biofunctional coatings have already seen applications in promoting endothelialization and cell growth,23-24 the capture and detection of circulating tumor cells,25 and protein-based nanotubes for the selective capture of nanoparticles, proteins and viruses.22 The detection of dilute protein biomarkers can facilitate early disease diagnosis and consequently treatment, thereby improving patient survival.26 An example of this potential is with prostate specific antigen (PSA). PSA is a blood biomarker secreted by normal and tumor cells27-28 with a concentration of ~ 4 ng/mL in serum28 having been attributed to the possible presence of prostate cancer. However, the detection of PSA alone is not decisive for a cancer diagnosis,29 nor can the PSA concentration be correlated with tumor size.30 Although the detection of PSA in serum must be combined with examination and a prostate biopsy, the detection of low concentrations of PSA remains widely studied for cancer detection and may be an effective tool for early diagnosis. Lab miniaturization could replace conventional protein detection techniques with more cost effective and higher throughput techniques. This has led to increased efforts to fabricate such point-of-care diagnosis devices.26, immunosorbent
assays,
or
31
other
Although gold standard techniques like enzyme-linked viable
approaches
such
as
those
based
on
electrochemiluminescence, electrophoresis, or proteomics have acceptable sensitivities, they typically require user expertise and may be prohibitively costly for widespread use. One promising example is the electrochemical detection of proteins, which usually uses an EDC-NHS amidification to anchor specific antibodies to a material surface.32-33 Such devices have been 3 ACS Paragon Plus Environment
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recently investigated in the capture of important biomarkers like interleukin-6 and prostate specific antigen.33-34 Herein, we studied the parameters that govern the availability of amine functional groups in PEM films for functionalization and PSA detection. Although there are numerous examples of PEM films functionalized post-assembly via the use of free carboxylic acid groups,35-42 the creation and functionalization of reactable amine groups is much less explored due to the difficulty of producing multilayer films with free, non-polymer anion bound ammonium groups.38 Thus, we explored and optimized parameters for maximizing amine functionalization in a well studied multilayer film architecture of poly(allylamine hydrochloride)/poly(styrene sulfonate), PAH/SPS.43-50 We applied optimal film parameters for the creation of biotinylated multilayers for the capture and detection of PSA protein, by using Qdots as probes.51 The resultant system presents a good sensitivity for PSA detection that is within the range of 0.4 ng/mL to 100 ng/mL.27, 52-53
Experimental Section Material: PAH: Poly(allylamine hydrochloride), 56,000 g/mol or 15,000 g/mol (Aldrich). SPS: Poly(styrene sulfonate), 70,000 g/mol (Sigma-Aldrich). Fluor: Fluorescein sodium salt (Sigma), NHS-Fluor: N-hydroxysuccinimide ester activated fluorescein (Pierce Biotechnology); NHSBiotin: (+)-Biotin N-hydroxysuccinimide ester (Sigma Aldrich); STA: Streptavidin (Pierce Biotechnology); Neutravidin protein (Pierce Biotechnology); Qdot® 655 Streptavidin Conjugate (Life Technologies); BSA: Albumin from bovine serum (Sigma Aldrich); PSAab: Monoclonal Antibody against Prostate-specific antigen, Biotin conjugate (Pierce Biotechnology); PSAag:
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Prostate Specific Antigen, human (Millipore); PBS: Phosphate-Buffered Saline (Corning Cellgro); FBS: fetal bovine serum (Life Technologies). LbL assembly: Polymer solution concentrations were 10 mM (based on repeat unit) with either 0 or 0.1 M NaCl. Polymers and deionized water rinse solutions were adjusted to desired pH using 1 M HCl or 1 M NaOH solutions. The PAH/SPS system was assembled at either pH 3.0 or 9.3 using a spin-assisted dipper StratoSequence VI, NanoStrata Inc. Glass slides or Si wafer were used as substrates and dipped into polymer solutions until the desired number of bilayers was reached (15.5 or 5.5 bilayers). The substrate was dipped for 10 minutes for the polymer solution followed by three rinse steps of 2, 2 and 1 minute each. Rinse water was adjusted to the same pH of the polymer solutions and no salt was added (except for the experiments using salt in all steps, where 0.1 M NaCl was added). One bilayer is comprised of one polycation (PAH) and one polyanion (SPS) layer. The polycation was assembled as the first and last layer, to increase the amine groups on the film surface. Films were used as deposited (AD) or after performing a postassembly acid treatment (AT) exposure to pH 2.5 for 15 min. Samples were rinsed twice in deionized water to remove any acid traces and dried with compressed air. All films used in this work were used within 10 hours after assembly. Determination of thicknesses in dry and wet state: Thicknesses and swelling degree of the PEMs films were measured using a spectroscopic ellipsometer (Woollam M-2000D; ISN, MIT). All thickness measurements were made at three different points on each film and averaged. For the swelling measurements, films were placed inside a fluid chamber.54 Note that in this case, the swelling measurements were taken in deionized water at pH 7 and in PBS for as-deposited or for pH treated (pH 2.5 or pH 10), rinsed and dried films. First, the pH treated rinsed and dried film thicknesses were measured. Then, the desired solution (deionized water or PBS) was added into 5 ACS Paragon Plus Environment
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the fluid chamber. After 5 minutes the swollen measurement was taken (we observed that after 5 minutes the swelling stabilized for all films). The percentage of swelling of the multilayers films can be defined as: % Swelling = 100(Tin solution – Tdry) / Tdry
(1)
Where Tdry is the thickness of the dried film and Tin solution is the film thickness after swelling. Post-assembly NHS functionalization: NHS-esters (NHS-Biotin and NHS-Fluor) were diluted into aliquots in DMSO (stock solution 20 mM) and stored at -20 °C. Prior to analyses the aliquots were thawed at room temperature and further diluted to 5 mM in PBS buffer. LbL films were reacted for 2 hours at room temperature, and then rinsed twice in PBS buffer and air-dried. To assess the reaction efficiency, we reacted NHS-Fluorescein (NHS-Fluor; 5 mM in PBS) to the different surfaces using Fluorescein sodium salt (Fluor; 5 mM in PBS) solution as a control for non-specific adsorption. After the reaction, we looked at the absorbance spectrum of the samples via UV-Vis spectroscopy. UV-vis spectra were acquired in the range of 450 to 600 nm (Varian Cary 5E spectrophotometer); an average of 4 samples were used. All solutions were adjusted to desired pH using 1 M NaOH solution before reaction. The absorbance data at 540 nm was averaged for each sample. For statistical analyses, t-tests with 95% confidence interval were performed using Prism 5 software. X-ray Photoelectron Spectroscopy (XPS): XPS was performed to determine the amount of amine and ammonium groups in the films. Chemical composition of approximately the top 10nm of the film was characterized using a PHI VersaProbe II X-ray photoelectron spectrometer with a scanning monochromated Al source (1,486.6 eV; 50 W; spot size, 200 μm). The takeoff angle between the sample surface and analyzer was 45°, and the X-ray beam collected C1s, N1s, O1s,
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and Si2p elemental information while rastering over a 200Χ700-μm area. Peak fitting analysis was done in CasaXPS. Qdots capture by biotinylated films: In order to simulate the capture of small biological particles (e.g. proteins), a validation experiment was performed using streptavidin coated Qdots. Films reacted with NHS-Biotin (pre or post assembled) were incubated with 20 nM of Qdots diluted in blocking buffer (PBS containing 6% of BSA). For control samples as deposited (AD) and acid treated (AT) reacted films were used. All samples were incubated with blocking buffer for 1h followed by two rinse steps in PBS, before reaction with Qdots. The use of blocking buffer was crucial to avoid the adsorption of Qdots by non-specific interactions on nonbiotinylated films. Streptavidin conjugated Qdots (STA-Qdots) were reacted for 2h at RT and rinsed twice in PBS buffer. Samples were observed in a fluorescence microscope, and the fluorescence intensity was quantified with Image J for each sample. An average of 3 experiments was used. PSA capture: Biotinylated films were incubated with blocking buffer for 1h, rinsed three times in PBS and then reacted with Neutravidin (50 µg/mL, 1 h, 4 ºC). Of note, we have examined many different conditions until reaching the best parameters of the current capture protocol. We found that using the blocking buffer prior to incubation of both neutravidin and PSA antigen helped prevent non-specific adsorption of the subsequent reagents. Three rinse steps in PBS were performed to remove unreacted Neutravidin. A solution of PSA biotinylated antibody (4 µg/mL) was reacted for 30 min, followed by three rinse steps in PBS. Another step of blocking buffer was performed previous to PSA capture in order to prevent nonspecific interactions. PSA antigen at desired concentration (100 ng/mL to 0.1 ng/mL) was diluted in either PBS buffer or fetal bovine serum (FBS, passed through 0.2 µm filters prior dilution of PSA), and then incubated for 7 ACS Paragon Plus Environment
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2 minutes followed by three rinse steps in PBS. PSA biotinylated antibody was reacted to the PSA antigen for 2 minutes followed by three rinse steps in PBS. Finally, 20 nM of streptavidin conjugated Qdots were incubated for 5 minutes to react with the biotinylated antibody. Samples were rinsed thrice in PBS and examined in a fluorescence microscope. The fluorescence intensity was quantified with Image J software. An average of 3 experiments was used. SEM was performed on 100 ng/mL of reacted antigen for biotinylated acid treated samples and on nonbiotinylated samples (control). Samples were coated with 5 ng of carbon before imaging.
Results and discussion Effects of molecular weight, ionic strength and film post treatment on film functionalization For the detection of the biomarker PSA at dilute concentrations in solution, it is important to maximize the ability for its capture. One way to achieve this is to substantially increase the density of PSA antibodies present within the PEM film. Thus, the first step in our investigation was to study the parameters related to reactive amine availability in a PEM film system with regards to functionalization for the capture of dilute antigens in solution. Using a PAH/SPS 9.3/9.3 system, the polycation poly(allylamine hydrochloride) (PAH, Figure 1) presents free primary amine groups that can be modified through simple amide-forming chemistry with a variety of reagents. PAH is a weak polycation with a pKa ~ 8.855 and by tuning the pH of assembly one can adjust its charge density, leading to fine control over the thickness of each deposited layer.56-57 As previously demonstrated by our group,44 PAH assembled with the strong polyelectrolyte poly(styrene sulfonate) (SPS, Figure 1) at pH 9.3 combines a partially ionized polycation with a fully ionized polyanion generating a film in which the non-ionized portions of PAH preferentially organize into hydrophobic pockets. When undergoing acid treatment (AT) by 8 ACS Paragon Plus Environment
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incubation in solutions of pH ≤ 4 for 15 minutes, films of the PAH/SPS 9.3/9.3 system demonstrate a high swelling capability that is not observed for the as deposited (AD) films. Acid treatment of the 9.3/9.3 films protonates the primary amines of PAH trapped in the hydrophobic pockets and causes their rearrangement within the film for greater solvent accessibility and consequently increased hydrophilicity that promotes additional swelling capability.44
(PAH)
(SPS)
Figure 1. Structural formulas of poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (SPS). In order to characterize the swelling capacity, the state of amine ionization and its influence on functionalizability, we evaluated a number of assembly parameters in the PAH/SPS 9.3/9.3 system and their effects on amine derivatization using N-hydroxysuccinimidyl ester (NHS) conjugation chemistry. Three different parameters were evaluated: the influence of ionic strength of solutions during assembly (no salt or 0.1 M NaCl), the molecular weight of PAH (15 or 56 kDa), and the effect of a post-assembly acid treatment. Growth curves (Figure S1, Table S1) for all multilayers tested were linear, giving a good control over the film thickness depending on the number of deposited bilayers. A detailed discussion regarding the parameters and their influence on the relative degrees of functionalization can be found in Supporting Information (Figure S2). Acid treated multilayer films were reacted with an NHS-ester of fluorescein, NHS-Fluor, and the resultant fluorescein
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absorbance was used as an indicator of the relative accessibilities of the primary amines. A summary of results can be found in Figure 2.
% of Increase in Fluorescein conjugation after acid treatment
40
PAH 15 kDa PAH 56 kDa 30
20
10
0
No salt
0.1 M NaCl in polymer solution only
0.1 M NaCl in polymer and rinse solutions
Sa lte dP oly /Ri ns e
Sa lte dP oly
Figure 2. Effect of PAH molecular weight and salt in rinse and/or polymer solutions on NHSNo Sa lt
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functionalization (performed at pH 8.3 for 2h) as measured by fluorescein conjugation after different assembly conditions. Data acquired by UV-Vis absorbance at 540 nm.
Fluorescein conjugation increased after acid treatment (AT) for all assembly ionic strength parameters (p < 0.05) as compared to as-deposited films (AD) films. This enhancement of fluorescein conjugation corresponds with the increased accessibility of free amines that is generated after acid treatment.44 Most notably, acid treatment of multilayer films assembled with salt and especially salt in the polymer solutions only resulted in significantly greater amounts of amine fluorescein functionalization, as observed in Figure 2 (See detailed discussion in Supporting Information, Figure S2). Previous studies by us with this multilayer system did not include salt in the polymer solutions. The addition of salt in the polymer assembly solutions clearly improves the functionalization capacity. In addition, by increasing the molecular weight of PAH from 15 kDa to 56 kDa, amine functionalization is improved (Figure S2). Previous 10 ACS Paragon Plus Environment
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works have shown that higher molecular weight polymers tend to arrange with a larger fraction of segmental loops when compared to shorter polymers, and therefore promote larger thickness increments per deposited bilayer.58-59 In this case, we hypothesize that the more loopy molecular organization created with higher molecular weight PAH facilitates more amine enriched hydrophobic pockets compared to the lower molecular weight PAH. To further investigate the parameters surrounding conjugation of the available amines, we varied the pH during fluorescein conjugation of films assembled with the optimal conditions identified above: high molecular weight PAH (56kDa), and salt in polymer solutions (no salt in rinse solutions). 0.20
pH 8.3 Reaction
pH 9 Reaction
pH 10 Reaction
Absorbance (a.u.)
0.18 0.16
*
0.14 0.12 0.10 0.08 0.06 0.04
A AD D N Flu H S- or Fl u A AT or T F N l H uo S- r Fl u A AD or D F N l H uo Sr Fl u A AT or T N Flu H S- or Fl u A AD or D F N l H uo Sr Fl u o r A AT T N Flu H S- or Fl uo r
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Figure 3. Effect of pH of conjugation reaction on PAH/SPS 9.3/9.3 15.5 bilayer films (PAH: 56 kDa / SPS: 70 kDa). All films assembled with 0.1 M NaCl in polymer solutions. As controls to determine non-specific interactions, films were treated under the same conditions with fluorescein sodium salt (Fluor) at 5mM for 2 h at room temperature. Absorbance at 540 nm was acquired by UV-vis. AD: as deposited films; AT: acid treated films. 11 ACS Paragon Plus Environment
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As observed in Figure 3, when fluorescein conjugation is performed with solution pHs in the range of 8 to 10, there is a greater degree of functionalization of the acid treated films than that of as-assembled films and the control samples: with pH 10 providing the highest level of functionalization. This improvement in functionalization suggests that there is a larger fraction of free amines present in their more reactive, non-ionized (and hence more nucleophilic) form. As noted above, this functionalization was significantly greater than that obtained by an NHSFluor reaction of as-assembled films, as well as the AD and AT films incubated with Fluor, which was used as a control for determining non-specific interactions.
Surface Composition of PAH/SPS films on different pH environments To further evaluate the surface composition of PAH/SPS films, we used X-ray Photoelectron Spectroscopy (XPS) to determine the ratio of amine and ammonium groups resulting from differences in pH of film deposition (pH 3.0 or 9.3) and post-assembly pH treatments. This technique allows for analysis of the elemental sample composition in the realm of 0-10 nm beneath the surface. Analysis was performed on samples fabricated with high molecular weight PAH (56kDa), and salt in polymer solutions (no salt in rinse solutions).
Table 1. Ratio of amine/ammonium groups available on film surfaces. Data obtained by XPS analyses (See Figure S3 for fitting example).
Sample (PAH3.0/SPS3.0)15.5
Amine (%)
Ammonium (%)
21
79 12
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(PAH9.3/SPS9.3)15.5 as deposited
44
56
(PAH9.3/SPS9.3)15.5 acid treated (pH 2.5, 15 min)
20
80
(PAH9.3/SPS9.3)15.5 acid treated + incubation at pH 8.3 for 2h
21
79
(PAH9.3/SPS9.3)15.5 acid treated + incubation at pH 9 for 2h
44
56
(PAH9.3/SPS9.3)15.5 acid treated + incubation at pH 10 for 2h
46
54
As detailed in Table 1, the as deposited 9.3/9.3 films contain about 56% of nitrogen groups present as ammonium groups. Subsequent acid treatment induces an increase in the ammonium fraction to 80%, as contact with pH 2.5 shifts the equilibrium towards protonation of the free nitrogen groups, including those contained in hydrophobic clusters. Taking the acid treated film and incubating it for 2h in pH 8.3 does not substantially affect the fraction of ammonium groups (79%), but incubation in pH 9 or 10 causes a significant shift of nitrogen groups into non-ionized amines. In previous infrared spectroscopy examinations of PAH9.3/SPS9.3 multilayer films assembled without salt,43 we observed slightly greater degrees of ionization for the nitrogen groups of PAH in as deposited films (70%),43 and acid treated films (90%). These slight differences may be due to salt shielding resulting in a smaller fraction of the total nitrogen groups of PAH to participate in electrostatic crosslinks with SPS within the film. The tendency for nitrogen groups participating in such electrostatic crosslinks to remain ionized beyond pH conditions that would deprotonate otherwise free nitrogen groups, could reflect the greater presence of ammonium groups in the previous salt-free films.60 Also, one should note that this difference might be observed as XPS analyzes the near-surface (
