Reduction of Non-Specific Protein Adsorption Using Poly(ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels In Immunoassays for Staphylococcal Enterotoxin B Detection

Three PEG molecules (PEG-methacrylate, -diacrylate and -dimethacrylate) were incorporated into galactose-based polyacrylate hydrogels and their relative abilities to reduce non-specific protein adsorption in immunoassays were determined. Highly crosslinked hydrogels containing amine-terminated functionalities were formed and used to covalently attach antibodies specific for staphylococcal enterotoxin B (SEB). Patterned arrays of immobilized antibodies in the PEG-modified hydrogels were created with a PDMS template containing micro-channels for use in sandwich immunoassays to detect SEB. Different concentrations of the toxin were applied to the hydrogel arrays, followed with a Cy3-labeled tracer antibody specific for the two toxins. Fluorescence laser scanning confocal microscopy of the tracer molecules provided both qualitative and quantitative measurements on the detection sensitivity and the reduction in non-specific binding as a result of PEG incorporation. Results showed the PEG-modified hydrogel significantly reduced non-specific protein binding with a detection limit for SEB of 1 ng/mL. Fluorescence signals showed a 10-fold decrease in the non-specific binding and a 6-fold increase in specific binding of SEB

Polyacrylamide hydrogel grafted onto polyethylene terephthalate textile via ultraviolet light-emitting diode photografting for oil/water separation

This study was invented to provide a cheaper alternative filter material for oil/water separation application. Polyethylene terephthalate (PET) textiles with diameter of 4.5 cm were chemically grafted with a thin layer of polyacrylamide (PAAm) hydrogel via UV LED photopolymerisation system. From the grammages that were tested, 85 gsm PET was the most favourable to be used as a matrix. Based on the weight loss data the alkali treatment was optimised under the following condition i.e. 2 hours of treatment with 10 wt % of NaOH at 60 ºC. For the grafting process, the effects of UV curing time (5-30 minutes), positioning of filter paper as a spacer (M1 representing the filter paper at the bottom side only and M2 representing the filter papers at both top and bottom sides) as well as technique of grafting (immersion and dipping) were also taken into the account. The obtained samples were characterised using the basic characteristics such as the degree of grafting (DG) and Fourier transformed infrared spectroscopy (FTIR). The DG values for immersed samples were significantly higher (190 % for M1 and 160 % for M2) than dipped samples (90 % for M1 and 60 % for M2). The obtained samples were also characterised in terms of surface morphology by field emission scanning electron microscopy (FESEM), oil fouling, pure water permeability and oil/water permeability test. The results indicated that, the oil/water separation performance of the hydrogel-grafted filter materials (PAAm-g-HPET) were strongly influenced by the DG of grafted PAAm hydrogel. However, the immersion grafting technique was found not suitable to be used for commercialisation purposes because of the low water permeability due to hydrogel grafting inside pores as evidenced by FESEM images. The dipping grafting method with the positioning of M2 was selected to be the best method to deal with filtration in oil/water separation. Different UV curing time influenced the oil fouling behaviour of filter samples. Data concluded that 20 minutes of curing was the optimum time for hydrogel grafting. Wettability data indicated that the filter materials after undergoing alkali treatment as well as after being grafted with PAAm hydrogel changed from hydrophobic to hydrophilic. To some extent, this innovation has shown in the near future as promising device for oil/water separation

Development of Immunoassay Using Graphene and Microfluidic Platforms.

Protein, as one of the most important functional biomolecules in the human body, plays a significant role in physiological responses and molecular diagnostics. Detecting the existence of proteins, quantifying concentration, and identifying protein types are therefore important techniques in many fields. Immunoassays are one of the major techniques relied on for protein detection. Immunoassays have been broadly applied in disease diagnosis, pharmaceutical development, food science, and environmental protection. The first part of this dissertation describes studies aimed at developing chemical vapor deposition (CVD) graphene as a large size protein biosensing platform. To utilize graphene as a biosensing platform, techniques to immobilize proteins on graphene are critical. In this dissertation work, carboxyl functional groups (-COOH) were created by graphene functionalization, and the functionalized graphene was characterized using Raman spectroscopy, X-ray photo spectroscopy (XPS), and fluorescence microscopy. The approach developed here provides information about protein coupling density and uniformity on large scale graphene (> cm2). The second and the third parts of the thesis describe the application of a microfluidic technique to two widely used protein detection methods – immunoblotting and dot blotting. The microfluidic systems were designed and fabricated to be easily interfaced with a common type of protein blotting membrane called polyvinylidene fluoride (PVDF) membrane. The microfluidic device was specifically applied to the antibody incubation step, which reduces antibody consumption and therefore also significantly reduces the cost of the assay. In microfluidic immunoblotting, an approach to activate the PVDF membrane to increase its protein binding capacity was developed. This was achieved by adding a surfactant Tween-20 to the antibody solution. The concentration of Tween-20 was optimized so that only the portion of the membrane within the channel region was activated. The system has been shown to be able to profile inflammatory signaling pathways. In microfluidic dot blotting, the influence of substrate hydrophobicity and protein concentrations on device design constraints were studied. Inflammatory cytokine detection using the developed microfluidic dot blotting system was determined. Altogether these experiments demonstrate that applying microfluidic techniques to protein immunoblotting and dot blotting improves detection efficiency, and reduces cost by utilizing less antibodies.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113501/1/huaining_1.pd

Kuvioidut ja bioaktiiviset nanoselluloosafilmit

In this thesis, fluidic channels were prepared on films made from cellulose nanofibers (CNF) and their potential use in biosensor applications was studied. The main goal was to develop hydrophilic-hydrophobic patterns to controllably produce CNF substrates for microfluidic applications. The work included a detailed investigation, to prevent non-specific adsorption of a type of human serum protein, hIgG, on CNF. A suitable antifouling agent for the CNF films was tested. CNF is a cellulosic material that has at least one dimension in the nanometer range and it is mainly produced mechanically from wood fibers. It can be used to make strong, translucent and smooth films. Two different approaches were tested to prepare 2D-channels on the CNF films: photolithography and inkjet printing of hydrophobic materials. The photolithographic method utilized simultaneously thiol-ene and thiol-yne click chemistries. In the inkjet studies, it was observed that polystyrene dissolved in p-xylene worked successfully. The prepared microfluidic CNF materials were characterized with SEM, AFM, contact angle measurements and liquid flow tests. Additionally, the non-specific protein adsorption was studied by using model CNF films with QCM-D, SPR and AFM techniques. Furthermore, the adsorption of fluorescent hIgG was performed on real CNF films and channels with CLSM method. The molecules used for protein blocking included BSA, fibrinogen and PDMAEMA-block-POEGMA copolymers. The results indicated that the best fluid flow was obtained by inkjet printing channels with polystyrene edges on CNF films. In addition, the PDMAEMA-block-POEGMA copolymer was the best antifouling agent for CNF and it reduced the hIgG adsorption up to 95 %. The successful blocking of the channels point out that these systems could be developed further and possibly be used in future biosensing applications.Tässä diplomityössä valmistettiin nestevirtauskanavia nanoselluloosa-filmien pinnoille, ja tutkittiin näiden mahdollista käyttöä biosensoreissa. Työn tavoitteena oli valmistaa kanavia muodostamalla filmeille hydrofobisia ja hydrofiilisiä alueita. Lisäksi tavoitteena oli tutkia hIgG-vasta-aineen epäspesifistä adsorptiota CNF-filmeille sekä löytää sopivia adsorptionestoaineita, jotka soveltuvat tälle materiaalille. Adsorptionestoaineina kokeiltiin BSA:ta, fibrinogeenia ja PDMAEMA-blokki-POEGMA -kopolymeerejä. Selluloosan nanofibrillit (cellulose nanofibrils CNF) ovat nanomateriaali, jota valmistetaan puukuiduista mekaanisella käsittelyllä. Tästä materiaalista voidaan valmistaa mekaanisesti vahvoja, läpinäkyviä ja tasaisia filmejä. Tässä työssä valmistettiin CNF-filmeille kanavia käyttäen CLICK-kemian reaktioita sekä polymeerikuviointi-menetelmää käyttäen mustesuihkutulostusta. Valmistettuja pintakanavia tutkittiin SEM- ja AFM-menetelmillä, kontaktikulmamittauksilla sekä virtaustestein. Proteiinien epäspesifistä adsorptiota tutkittiin CNF-mallipinnoilla QCM-D-, SPR- ja AFM-menetelmien avulla. Lisäksi adsorptiota tutkittiin CNF filmeille valmistetuissa kanavissa fluoresoidun vasta-aineen avulla CLSM-menetelmällä. Tutkimukset osoittivat, että mustesuihkutulostettu polystyreeniliuos muodosti parhaiten toimivan nestevirtauskanavan CNF filmin pinnalle. Lisäksi PDMAEMA-blokki-POEGMA -kopolymeeri osoittautui parhaaksi adsorptionestoaineeksi. Se vähensi hIgG:n adsorptiota 95 %. Yhdistämällä pintakanavien valmistusmenetelmä ei-selektiivisen adsorptionestoaineiden kanssa voidaan valmistaa materiaaleja tulevaisuuden biosensoreihin

Laser-Assisted Surface Modification of Hybrid Hydrogels to Prevent Bacterial Contamination and Protein Fouling

Silicone hydrogels have been extensively studied in the fields of contact lenses, tissue engineering, and drug delivery due to their good biocompatibility, high oxygen permeability, and proper light transmission. However, their applications in biomedical devices are limited by protein adsorption and bacterial contamination because of the hydrophobic surface of silicone, which will cause more irreversible protein adsorption. Several physical methods can be applied to create a hydrophilic surface on hydrogels, such as spin coating, physical vapor deposition, dip coating, drop casting, etc. Compared to the conventional methods, the matrix assisted pulsed laser evaporation (MAPLE) is suitable to produce biopolymer/polymer film with a contamination-free manner. In this thesis, hydrophilic polymer, polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP), were deposited by MAPLE with a pulsed Nd:YAG 532 nm laser for the surface hydrophilicity modification. The polymer coatings were characterized by Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Our results demonstrate that protein adsorption decreases 28.2% and 18.7% with the surface modifications by PEG and PVP, respectively. In addition, the polymer coated silicone hydrogels do not impose toxic effect on mouse NIH/3T3 cells. Normally, protein fouling can lead to biofilm contamination caused by the growth of bacteria. Therefore, we further deposit hybrid nanocomposite on silicone hydrogels to inhibit the growth of bacteria. Silver nanoparticles incorporating with PVP (Ag-PVP NPs) were developed through a photochemical method without addition of reductive reagents. On the other hand, sol-gel method was applied to incorporate ZnO nanoparticles into PEG (ZnO-PEG NPs). MAPLE process was applied to deposit the two different nanocomposites on the silicone hydrogels, respectively. Our results indicate that the silicone hydrogels with Ag-PVP nanocomposite coating can reduce 28.2% of the protein adsorption compared to silicone hydrogels without coating, while ZnO-PEG coating is able to reduce 30% protein adsorption. The cytotoxicity study shows that the nanocomposite coated silicone hydrogels do not impose toxic effect on mouse NIH/3T3 cells. In addition, MAPLE-deposited Ag-PVP and ZnO-PEG nanocomposite coatings can inhibit bacterial growth significantly. Our result show that Ag-PVP nanocomposite coating can eliminate almost all the E.coli after 8 hours’ culturing; the relative numbers of E.coli on the ZnO-PEG coated silicone hydrogel approach to zero when the culturing time is 4 hours. In addition, the thickness and roughness of Ag-PVP film over time were measured by AFM. The result shows that MAPLE process is a time dependent (linear) deposition, and it is able to create homogenous thin films (roughness is lower than 30 nm). MAPLE shows good ability to control the thickness in the deposition of organic molecules and nanoparticles, which maintains the chemical backbone of polymers, and prevents contamination

Modification of Self-Assembled Monolayers and Hydrogel Nanomembranes by Ultraviolet Light

Custom design of organic and biological surfaces and soft matter lithography are important issues of modern nanotechnology and physical chemistry of interfaces. An important tool in this regard is ultraviolet (UV) light which can be used for controlled modification and patterning of organic and biological surfaces. In this context, the effect of UV light on alkanethiolate (AT) self-assembled monolayers (SAMs) on gold substrates was studied, with a particular emphasis on its wavelength dependence. The experiments were first performed for the most basic system of non-substituted AT SAMs which exhibited qualitatively similar photooxidation behavior at UV wavelength variation from 254 to 375 nm but a strong decrease of the photooxidation cross-section with increasing wavelength. Based on these results, the possibility of UV-promoted exchange reaction (UVPER) with non-substituted AT SAMs as the primary matrix and azide-substituted ATs as substituents was tested and successfully realized, resulting in the fabrication of mixed SAMs with variable density of the azide tail groups, capable of the subsequent click reaction with various kinds of molecules and functional moieties with alkynyl group. Such a click reaction with several representative substituents was demonstrated. Further, the above approach was extended to oligo(ethylen glycole) substituted AT SAMs serving as protein repelling primary matrix. Combining UVPER and the subsequent click reaction with a biotin-bearing substituent, biorepulsive templates with controlled density of the docking sites for the specific adsorption of biotin-complementary proteins such as avidin and streptavidin were prepared and successfully tested regarding their non-specific and specific protein affinity. This approach was extended to UV lithography, resulting in preparation of custom-designed, gradient protein-adhesion patterns. Finally, based on the results for the OEG-AT SAMs, the effect of UV light on protein-repelling poly(ethylen glycole) (PEG) nanomembranes was studied. It was demonstrated that UV irradiation induces extensive desorption of the PEG material, without photooxidation or other noticeable changes in the chemical composition, biorepelling behavior and hydrogel properties of the residual membrane. This opens a new way of 3D patterning of all-PEG materials, potentially useful for nanofabrication and biotechnology

Influencing the Inflammatory Response Through Multi-Scale Geometry, Antibiotic Release, and Fluid Management in a Textile-Based Biomaterial Wound Dressing

The total population of diagnosed and undiagnosed diabetes mellitus in the United states is expected to rise by 54% between the years of 2015 and 2030 contributing to $200 billion in health care expense. The exponential rise in common diabetic wounds, such as diabetic foot ulcers, puts a large population at risk for complications such as infection, amputation, and even death. Peripheral neuropathy leading to late diagnoses, patient non-compliance, and lack of holistic treatment options all contribute to complications with the incidence of new ulcer formation after treatment reaching 50%. This work explores the design, development, and in vitro evaluation of a multicomponent textile-based biomaterial and absorptive dressing that combines the need to manage infection, eliminate excess exudate levels, and provide an ideal environment for healthy tissue to repair and remodel the wound site. Melt-splun poly-l-lactide (PLLA) yarn of fibers with round or 4-deep-grooved (4DG) geometry were knitted into the skin-contact layer, the first layer of the dressing. Different methods of gentamicin sulfate (GS) incorporation, along with the impact of fiber geometry, were studied to explore optimal antibiotic release and efficacy. Results indicated that an increase in surface area as well as heat-enabled diffusion allowed for higher release of GS. Because each factorial treatment, with the exception of exhaustion dyeing method of incorporation, released GS at or above the minimum inhibitory concentration, there showed no difference in geometry and method of incorporation on antibacterial efficacy. The GS incorporated skin contact layer also appeared to be biocompatible in cultures of mouse bone marrow stromal D1 cells. Cell adhesion studies showed that a polyethylene glycol (PEG) surface treatment is needed to prevent non-specific protein and cellular attachment upon dressing changes. A microscopically thin layer of PEG was added to the surface of the contact layer and showed less cell attachment as seen in fluorescently labeled LIVE/DEADTM analysis, while showing no impact on GS release and antibacterial efficacy. In this aim, it can be concluded that the combination of GS release and a PEG surface coat can simultaneously kill and prevent infection while providing a non-adhesive surface upon removal from the wound. Polyurethane (PU) foam was characterized in a two-factor analysis based on foam density and mixing speed used to create the foam layer. PU foam V was chosen as the absorptive layer of the dressing and a comparative analysis was conducted using commercialized absorptive dressings. The PU foam layer was exposed to different time durations of ultra-violet ozone to increase the surface wettability and initiate moisture absorption. To prevent saturation, PLLA yarn of 4DG fibers was braided into an evaporative and moisture wicking layer. The braided fabric was able to vertically wick porcine serum at a rate of 0.88 mm/sec. The combination of absorptive and moisture wicking layers stimulate wound healing by removing moisture from the ulcer, while preventing maceration and premature saturation of the dressing, leading to fewer dressing changes. Additionally, an in vitro chronic wound model was constructed to verify the efficacy of the combined layers of the dressing. After applying the dressing for a duration of 48 hours, the dressing inhibited bacterial infection, while acting as a superabsorbent material without causing saturation. Further work explored healthy cell viability and any oxidative stress levels after exposing cells to both bacterial infection and the dressing. Although the in vitro model maintains some limitations and assumptions at the present time, it can be concluded that with the addition of the wound dressing, cell viability increased over time, and therefore promoted tissue repair. Future work will explore alternative antimicrobials for a more gradual release as well as improving the in vitro model by discovering the interaction between the co-culture in different types of medias and substrates while including proinflammatory biomarkers that could affect oxidative stress

Surface-Enhanced Raman Spectroscopy-Based Biomarker Detection for B-Cell Malignancies

This thesis presents a light scattering-based method for biomarker detection, which could potentially be used for the quantification of multiple biomarkers specific to B-cell malignancies. This method uses fabricated gold nanoparticle probes to amplify inelastic light scattering in a process referred to as surface-enhanced Raman scattering. These gold nanoparticle probes were conjugated to antibodies for specific and targeted molecular binding. The spectrum of the amplified inelastic light scattering was detected using a spectrometer and a detector. To detect the light scattering signal from the gold nanoparticle probes, several commercial Raman spectrometer instruments were evaluated. Initial results from these evaluations are presented in this thesis. After system evaluation, a custom Raman microscope system was designed, built, and tested. This system was used for the development of a surface-enhanced Raman spectroscopy-based immunoassay. The development of this assay confirms the successful design of gold nanoparticle probes for the specific targeting and detection of immunoglobulins. The immunoassay also shows promise for the simultaneous detection of multiple biomarkers specific to B-cell malignancies