Reproducible and label-free biosensor for the selective extraction and rapid detection of proteins in biological fluids

Erythropoietin (EPO), a glycoprotein hormone of ∼34 kDa, is an important hematopoietic growth factor, mainly produced in the kidney and controls the number of red blood cells circulating in the blood stream. Sensitive and rapid recombinant human EPO (rHuEPO) detection tools that improve on the current laborious EPO detection techniques are in high demand for both clinical and sports industry. A sensitive aptamer-functionalized biosensor (aptasensor) has been developed by controlled growth of gold nanostructures (AuNS) over a gold substrate (pAu/AuNS). The aptasensor selectively binds to rHuEPO and, therefore, was used to extract and detect the drug from horse plasma by surface enhanced Raman spectroscopy (SERS). Due to the nanogap separation between the nanostructures, the high population and distribution of hot spots on the pAu/AuNS substrate surface, strong signal enhancement was acquired. By using wide area illumination (WAI) setting for the Raman detection, a low RSD of 4.92% over 150 SERS measurements was achieved. The significant reproducibility of the new biosensor addresses the serious problem of SERS signal inconsistency that hampers the use of the technique in the field. The WAI setting is compatible with handheld Raman devices. Therefore, the new aptasensor can be used for the selective extraction of rHuEPO from biological fluids and subsequently screened with handheld Raman spectrometer for SERS based in-field protein detection.

Synthesis of an erodible biomimetic hydrogel for drug delivery using native chemical ligation

Hydrogels are hydrophilic, three dimensional polymers that imbibe large quantities of water while remaining insoluble in aqueous solutions due to chemical or physical cross-linking. The polymers swell in water or biological fluids, immobilizing the bioactive agent, leading to drug release in a well-defined specific manner. Thus the hydrogels’ elastic properties, swellability and biocompatibility make them excellent formulations for drug delivery. Currently, many drug potencies and therapeutic effects are limited or otherwise reduced because of the partial degradation that occurs before the administered drug reaches the desired site of action. On the other hand, sustained release medications release drugs continually, rather than providing relief of symptoms and protection solely when necessary. In fact, it would be much better if drugs could be administered in a manner that precisely matches physiological needs at desired times and at the desired site (site specific targeting). There is therefore an unmet need to develop controlled drug delivery systems especially for delivery of peptide and protein bound drugs. The purpose of this project is to produce hydrogels for structural drug delivery and time-dependent sustained release of drugs (bioactive agents). We use an innovative polymerisation strategy based on native chemical ligation (NCL) to covalently cross-link polymers to form hydrogels. When mixed in aqueous solution, four armed (polyethylene glycol) amine (PEG-4A) end functionalised with thioester and four branched Nterminal cysteine peptide dendrimers spontaneously conjugated to produce biomimetic hydrogels. These hydrogels showed superior resistance to shear stress compared to an equivalent PEG macromonomer system and were shown to be proteolytically degradable with concomitant release of a model payload molecule. This is the first report of a peptide dendrimers/PEG macromonomer approach to hydrogel production and opens up the prospect of facile hydrogel synthesis together with tailored payload release.

Ultra-trace detection of diagnostically important biomarkers using functionalised-Surface Enhanced Raman Spectroscopy (SERS)

Here we report an ultrasensitive method for detecting bio-active compounds in biological samples by means of functionalised nanoparticles interrogated by surface enhanced Raman spectroscopy (SERS). This method is applicable to the recovery and detection of many diagnostically important peptidyl analytes such as insulin, human growth hormone, growth factors (IGFs) and erythropoietin (EPO), as well as many small molecule analytes and metabolites. Our method, developed to detect EPO, demonstrates its utility in a complex yet well defined biological system. Recombinant human EPO (rhEPO) and EPO analogues have successfully been used to treat anaemia in end-stage renal failure, chronic disorders and infections, cancer and AIDS. Current methods for EPO testing are lengthy, laborious and relatively insensitive to low concentrations. In our rapid screening methodology, gold nanoparticles were functionalised with anti-EPO antibodies to provide very high selectivity towards the EPO protein in urine. These “smart sensor” nanoparticles interact with and trap EPO. Subsequent SERS screening allows for the detection and quantisation of ultra trace amounts (<<10-15 M) of EPO in urine samples with minimal sample preparation. We present data showing that the SERS spectrum differentiates between human endogenous EPO and rhEPO in unpurified urine, and potentially distinguishes between purified EPO isoforms. The elimination of sample preparation and direct screening in biological fluids significantly reduces the time required by current methods. Antibody recognition against a variety of biological targets and the availability of portable commercial SERS analysers for rapid onsite testing suggest broad diagnostic applicability in a flexible analytical platform.

A novel identification method for ultra trace detection of biomolecules using functionalised Surface Enhanced Raman Spectroscopy (SERS)

This thesis developed a new method for measuring extremely low amounts of organic and biological molecules, using Surface enhanced Raman Spectroscopy. This method has many potential applications, e.g. medical diagnosis, public health, food provenance, antidoping, forensics and homeland security. The method development used caffeine as the small molecule example, and erythropoietin (EPO) as the large molecule. This method is much more sensitive and specific than currently used methods; rapid, simple and cost effective. The method can be used to detect target molecules in beverages and biological fluids without the usual preparation steps.

Nanostructured silver-gold bimetallic SERS substrates for selective identification of bacteria in human blood

Surface-enhanced Raman spectroscopy (SERS) is a potentially important tool in the rapid and accurate detection of pathogenic bacteria in biological fluids. However, for diagnostic application of this technique, it is necessary to develop a highly sensitive, stable, biocompatible and reproducible SERS-active substrate. In this work, we have developed a silver–gold bimetallic SERS surface by a simple potentiostatic electrodeposition of a thin gold layer on an electrochemically roughened nanoscopic silver substrate. The resultant substrate was very stable under atmospheric conditions and exhibited the strong Raman enhancement with the high reproducibility of the recorded SERS spectra of bacteria (E. coli, S. enterica, S. epidermidis, and B. megaterium). The coating of the antibiotic over the SERS substrate selectively captured bacteria from blood samples and also increased the Raman signal in contrast to the bare surface. Finally, we have utilized the antibiotic-coated hybrid surface to selectively identify different pathogenic bacteria, namely E. coli, S. enterica and S. epidermidis from blood samples.

Rapid isolation and detection of erythropoietin in blood plasma by magnetic core gold nanoparticles and portable Raman spectroscopy

Isolating, purifying, and identifying proteins in complex biological matrices is often difficult, time consuming, and unreliable. Herein we describe a rapid screening technique for proteins in biological matrices that combines selective protein isolation with direct surface enhanced Raman spectroscopy (SERS) detection. Magnetic core gold nanoparticles were synthesised, characterised, and subsequently functionalized with recombinant human erythropoietin (rHuEPO)-specific antibody. The functionalized nanoparticles were used to capture rHuEPO from horse blood plasma within 15 minutes. The selective binding between the protein and the functionalized nanoparticles was monitored by SERS. The purified protein was then released from the nanoparticles’ surface and directly spectroscopically identified on a commercial nanopillar SERS substrate. ELISA independently confirmed the SERS identification and quantified the released rHuEPO. Finally, the direct SERS detection of the extracted protein was successfully demonstrated for in-field screening by a handheld Raman spectrometer within 1 minute sample measurement time.

The current status of heart failure diagnostic biomarkers

Heart failure (HF) affects approximately 23 million individuals worldwide and this number is increasing, due to an aging and growing population. Early detection of HF is crucial in the management of this debilitating disease. Current diagnostic methods for HF rely heavily on clinical imaging techniques and blood analysis, which makes them less than ideal for population-based screening purposes. Studies focusing on developing novel biomarkers for HF have utilized various techniques and biological fluids, including urine and saliva. Promising results from these studies imply that these body fluids can be used in evaluating the clinical manifestation of HF and will one day be integrated into a clinical workflow and facilitate HF management.

The effects of bioactive akermanite on physiochemical, drug-delivery and biological properties of poly (lactide-co-glycolide) beads

Poly(lactide-co-glycolide) (PLGA) beads have been widely studied as a potential drug/protein carrier. The main shortcomings of PLGA beads are that they lack bioactivity and controllable drug-delivery ability, and their acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Akermanite (AK) (Ca(2) MgSi(2) O(7) ) is a novel bioactive ceramic which has shown excellent bioactivity and degradation in vivo. This study aimed to incorporate AK to PLGA beads to improve the physiochemical, drug-delivery, and biological properties of PLGA beads. The microstructure of beads was characterized by SEM. The effect of AK incorporating into PLGA beads on the mechanical strength, apatite-formation ability, the loading and release of BSA, and the proliferation, and differentiation of bone marrow stromal cells (BMSCs) was investigated. The results showed that the incorporation of AK into PLGA beads altered the anisotropic microporous structure into homogenous one and improved their compressive strength and apatite-formation ability in simulated body fluids (SBF). AK neutralized the acidic products from PLGA beads, leading to stable pH value of 7.4 in biological environment. AK led to a sustainable and controllable release of bovine serum albumin (BSA) in PLGA beads. The incorporation of AK into PLGA beads enhanced the proliferation and alkaline phosphatase activity of BMSCs. This study implies that the incorporation of AK into PLGA beads is a promising method to enhance their physiochemical and biological property. AK/PLGA composite beads are a potential bioactive drug-delivery system for bone tissue repair.

Improving behaviour classification consistency: a technique from biological taxonomy

Quantitative behaviour analysis requires the classification of behaviour to produce the basic data. In practice, much of this work will be performed by multiple observers, and maximising inter-observer consistency is of particular importance. Another discipline where consistency in classification is vital is biological taxonomy. A classification tool of great utility, the binary key, is designed to simplify the classification decision process and ensure consistent identification of proper categories. We show how this same decision-making tool - the binary key - can be used to promote consistency in the classification of behaviour. The construction of a binary key also ensures that the categories in which behaviour is classified are complete and non-overlapping. We discuss the general principles of design of binary keys, and illustrate their construction and use with a practical example from education research.

Development of a biaxial compression device for biological samples : preliminary experimental results for a closed cell foam

Biological tissues are subjected to complex loading states in vivo and in order to define constitutive equations that effectively simulate their mechanical behaviour under these loads, it is necessary to obtain data on the tissue's response to multiaxial loading. Single axis and shear testing of biological tissues is often carried out, but biaxial testing is less common. We sought to design and commission a biaxial compression testing device, capable of obtaining repeatable data for biological samples. The apparatus comprised a sealed stainless steel pressure vessel specifically designed such that a state of hydrostatic compression could be created on the test specimen while simultaneously unloading the sample along one axis with an equilibrating tensile pressure. Thus a state of equibiaxial compression was created perpendicular to the long axis of a rectangular sample. For the purpose of calibration and commissioning of the vessel, rectangular samples of closed cell ethylene vinyl acetate (EVA) foam were tested. Each sample was subjected to repeated loading, and nine separate biaxial experiments were carried out to a maximum pressure of 204 kPa (30 psi), with a relaxation time of two hours between them. Calibration testing demonstrated the force applied to the samples had a maximum error of 0.026 N (0.423% of maximum applied force). Under repeated loading, the foam sample demonstrated lower stiffness during the first load cycle. Following this cycle, an increased stiffness, repeatable response was observed with successive loading. While the experimental protocol was developed for EVA foam, preliminary results on this material suggest that this device may be capable of providing test data for biological tissue samples. The load response of the foam was characteristic of closed cell foams, with consolidation during the early loading cycles, then a repeatable load-displacement response upon repeated loading. The repeatability of the test results demonstrated the ability of the test device to provide reproducible test data and the low experimental error in the force demonstrated the reliability of the test data.

Biological performance of a polycaprolactone-based scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery

A bioactive and bioresorbable scaffold fabricated from medical grade poly (epsilon-caprolactone) and incorporating 20% beta-tricalcium phosphate (mPCL–TCP) was recently developed for bone regeneration at load bearing sites. In the present study, we aimed to evaluate bone ingrowth into mPCL–TCP in a large animal model of lumbar interbody fusion. Six pigs underwent a 2-level (L3/4; L5/6) anterior lumbar interbody fusion (ALIF) implanted with mPCL–TCP þ 0.6 mg rhBMP-2 as treatment group while four other pigs implanted with autogenous bone graft served as control. Computed tomographic scanning and histology revealed complete defect bridging in all (100%) specimen from the treatment group as early as 3 months. Histological evidence of continuing bone remodeling and maturation was observed at 6 months. In the control group, only partial bridging was observed at 3 months and only 50% of segments in this group showed complete defect bridging at 6 months. Furthermore, 25% of segments in the control group showed evidence of graft fracture, resorption and pseudoarthrosis. In contrast, no evidence of graft fractures, pseudoarthrosis or foreign body reaction was observed in the treatment group. These results reveal that mPCL–TCP scaffolds could act as bone graft substitutes by providing a suitable environment for bone regeneration in a dynamic load bearing setting such as in a porcine model of interbody spine fusion.