Improved production of industrially relevant recombinant proteins: application of multi-factorial design of experiments and scalable process modelling

The work described in this thesis focuses on the use of a design-of-experiments approach in a multi-well mini-bioreactor to enable the rapid establishments of high yielding production phase conditions in yeast, which is an increasingly popular host system in both academic and industrial laboratories. Using green fluorescent protein secreted from the yeast, Pichia pastoris, a scalable predictive model of protein yield per cell was derived from 13 sets of conditions each with three factors (temperature, pH and dissolved oxygen) at 3 levels and was directly transferable to a 7 L bioreactor. This was in clear contrast to the situation in shake flasks, where the process parameters cannot be tightly controlled. By further optimisating both the accumulation of cell density in batch and improving the fed-batch induction regime, additional yield improvement was found to be additive to the per cell yield of the model. A separate study also demonstrated that improving biomass improved product yield in a second yeast species, Saccharomyces cerevisiae. Investigations of cell wall hydrophobicity in high cell density P. pastoris cultures indicated that cell wall hydrophobin (protein) compositional changes with growth phase becoming more hydrophobic in log growth than in lag or stationary phases. This is possibly due to an increased occurrence of proteins associated with cell division. Finally, the modelling approach was validated in mammalian cells, showing its flexibility and robustness. In summary, the strategy presented in this thesis has the benefit of reducing process development time in recombinant protein production, directly from bench to bioreactor.

Handbook of research on user interface design and evaluation for mobile technology

In recent years, mobile technology has been one of the major growth areas in computing. Designing the user interface for mobile applications, however, is a very complex undertaking which is made even more challenging by the rapid technological developments in mobile hardware. Mobile human-computer interaction, unlike desktop-based interaction, must be cognizant of a variety of complex contextual factors affecting both users and technology. The Handbook of Research on User Interface Design and Evaluation provides students, researchers, educators, and practitioners with a compendium of research on the key issues surrounding the design and evaluation of mobile user interfaces, such as the physical environment and social context in which a mobile device is being used and the impact of multitasking behavior typically exhibited by mobile-device users. Compiling the expertise of over 150 leading experts from 26 countries, this exemplary reference tool will make an indispensable addition to every library collection.

Rapid testing for group B streptococcus during labour: a test accuracy study with evaluation of acceptability and cost-effectiveness

OBJECTIVE: To determine the accuracy, acceptability and cost-effectiveness of polymerase chain reaction (PCR) and optical immunoassay (OIA) rapid tests for maternal group B streptococcal (GBS) colonisation at labour. DESIGN: A test accuracy study was used to determine the accuracy of rapid tests for GBS colonisation of women in labour. Acceptability of testing to participants was evaluated through a questionnaire administered after delivery, and acceptability to staff through focus groups. A decision-analytic model was constructed to assess the cost-effectiveness of various screening strategies. SETTING: Two large obstetric units in the UK. PARTICIPANTS: Women booked for delivery at the participating units other than those electing for a Caesarean delivery. INTERVENTIONS: Vaginal and rectal swabs were obtained at the onset of labour and the results of vaginal and rectal PCR and OIA (index) tests were compared with the reference standard of enriched culture of combined vaginal and rectal swabs. MAIN OUTCOME MEASURES: The accuracy of the index tests, the relative accuracies of tests on vaginal and rectal swabs and whether test accuracy varied according to the presence or absence of maternal risk factors. RESULTS: PCR was significantly more accurate than OIA for the detection of maternal GBS colonisation. Combined vaginal or rectal swab index tests were more sensitive than either test considered individually [combined swab sensitivity for PCR 84% (95% CI 79-88%); vaginal swab 58% (52-64%); rectal swab 71% (66-76%)]. The highest sensitivity for PCR came at the cost of lower specificity [combined specificity 87% (95% CI 85-89%); vaginal swab 92% (90-94%); rectal swab 92% (90-93%)]. The sensitivity and specificity of rapid tests varied according to the presence or absence of maternal risk factors, but not consistently. PCR results were determinants of neonatal GBS colonisation, but maternal risk factors were not. Overall levels of acceptability for rapid testing amongst participants were high. Vaginal swabs were more acceptable than rectal swabs. South Asian women were least likely to have participated in the study and were less happy with the sampling procedure and with the prospect of rapid testing as part of routine care. Midwives were generally positive towards rapid testing but had concerns that it might lead to overtreatment and unnecessary interference in births. Modelling analysis revealed that the most cost-effective strategy was to provide routine intravenous antibiotic prophylaxis (IAP) to all women without screening. Removing this strategy, which is unlikely to be acceptable to most women and midwives, resulted in screening, based on a culture test at 35-37 weeks' gestation, with the provision of antibiotics to all women who screened positive being most cost-effective, assuming that all women in premature labour would receive IAP. The results were sensitive to very small increases in costs and changes in other assumptions. Screening using a rapid test was not cost-effective based on its current sensitivity, specificity and cost. CONCLUSIONS: Neither rapid test was sufficiently accurate to recommend it for routine use in clinical practice. IAP directed by screening with enriched culture at 35-37 weeks' gestation is likely to be the most acceptable cost-effective strategy, although it is premature to suggest the implementation of this strategy at present.

Using a systems approach in logistics design and planning

The international economic and business environment continues to develop at a rapid rate. Increasing interactions between economies, particularly between Europe and Asia, has raised many important issues regarding transport infrastructure, logistics and broader supply chain management. The potential exists to further stimulate trade provided that these issues are addressed in a logical and systematic manner. However, if this potential is to be realised in practice there is a need to re-evaluate current supply chain configurations. A mismatch currently exists between the technological capability and the supply chain or logistical reality. This mismatch has sharpened the focus on the need for robust approaches to supply chain re-engineering. Traditional approaches to business re-engineering have been based on manufacturing systems engineering and business process management. A recognition that all companies exist as part of bigger supply chains has fundamentally changed the focus of re-engineering. Inefficiencies anywhere in a supply chain result in the chain as a whole being unable to reach its true competitive potential. This reality, combined with the potentially radical impact on business and supply chain architectures of the technologies associated with electronic business, requires organisations to adopt innovative approaches to supply chain analysis and re-design. This paper introduces a systems approach to supply chain re-engineering which is aimed at addressing the challenges which the evolving business environment brings with it. The approach, which is based on work with a variety of both conventional and electronic supply chains, comprises underpinning principles, a methodology and guidelines on good working practice, as well as a suite of tools and techniques. The adoption of approaches such as that outlined in this paper helps to ensure that robust supply chains are designed and implemented in practice. This facilitates an integrated approach, with involvement of all key stakeholders throughout the design process.

Multi-frequency segmental bio-impedance device:design, development and applications

Bio-impedance analysis (BIA) provides a rapid, non-invasive technique for body composition estimation. BIA offers a convenient alternative to standard techniques such as MRI, CT scan or DEXA scan for selected types of body composition analysis. The accuracy of BIA is limited because it is an indirect method of composition analysis. It relies on linear relationships between measured impedance and morphological parameters such as height and weight to derive estimates. To overcome these underlying limitations of BIA, a multi-frequency segmental bio-impedance device was constructed through a series of iterative enhancements and improvements of existing BIA instrumentation. Key features of the design included an easy to construct current-source and compact PCB design. The final device was trialled with 22 human volunteers and measured impedance was compared against body composition estimates obtained by DEXA scan. This enabled the development of newer techniques to make BIA predictions. To add a ‘visual aspect’ to BIA, volunteers were scanned in 3D using an inexpensive scattered light gadget (Xbox Kinect controller) and 3D volumes of their limbs were compared with BIA measurements to further improve BIA predictions. A three-stage digital filtering scheme was also implemented to enable extraction of heart-rate data from recorded bio-electrical signals. Additionally modifications have been introduced to measure change in bio-impedance with motion, this could be adapted to further improve accuracy and veracity for limb composition analysis. The findings in this thesis aim to give new direction to the prediction of body composition using BIA. The design development and refinement applied to BIA in this research programme suggest new opportunities to enhance the accuracy and clinical utility of BIA for the prediction of body composition analysis. In particular, the use of bio-impedance to predict limb volumes which would provide an additional metric for body composition measurement and help distinguish between fat and muscle content.

Enabling long journeys in electric vehicles:design and demonstration of an infrastructure location model

This research develops a methodology and model formulation which suggests locations for rapid chargers to help assist infrastructure development and enable greater battery electric vehicle (BEV) usage. The model considers the likely travel patterns of BEVs and their subsequent charging demands across a large road network, where no prior candidate site information is required. Using a GIS-based methodology, polygons are constructed which represent the charging demand zones for particular routes across a real-world road network. The use of polygons allows the maximum number of charging combinations to be considered whilst limiting the input intensity needed for the model. Further polygons are added to represent deviation possibilities, meaning that placement of charge points away from the shortest path is possible, given a penalty function. A validation of the model is carried out by assessing the expected demand at current rapid charging locations and comparing to recorded empirical usage data. Results suggest that the developed model provides a good approximation to real world observations, and that for the provision of charging, location matters. The model is also implemented where no prior candidate site information is required. As such, locations are chosen based on the weighted overlay between several different routes where BEV journeys may be expected. In doing so many locations, or types of locations, could be compared against one another and then analysed in relation to siting practicalities, such as cost, land permission and infrastructure availability. Results show that efficient facility location, given numerous siting possibilities across a large road network can be achieved. Slight improvements to the standard greedy adding technique are made by adding combination weightings which aim to reward important long distance routes that require more than one charge to complete.

Nanotechnology and microfluidics:formulation design and on-chip manufacture of nanoparticles

Nanoparticles offer an ideal platform for the delivery of small molecule drugs, subunit vaccines and genetic constructs. Besides the necessity of a homogenous size distribution, defined loading efficiencies and reasonable production and development costs, one of the major bottlenecks in translating nanoparticles into clinical application is the need for rapid, robust and reproducible development techniques. Within this thesis, microfluidic methods were investigated for the manufacturing, drug or protein loading and purification of pharmaceutically relevant nanoparticles. Initially, methods to prepare small liposomes were evaluated and compared to a microfluidics-directed nanoprecipitation method. To support the implementation of statistical process control, design of experiment models aided the process robustness and validation for the methods investigated and gave an initial overview of the size ranges obtainable in each method whilst evaluating advantages and disadvantages of each method. The lab-on-a-chip system resulted in a high-throughput vesicle manufacturing, enabling a rapid process and a high degree of process control. To further investigate this method, cationic low transition temperature lipids, cationic bola-amphiphiles with delocalized charge centers, neutral lipids and polymers were used in the microfluidics-directed nanoprecipitation method to formulate vesicles. Whereas the total flow rate (TFR) and the ratio of solvent to aqueous stream (flow rate ratio, FRR) was shown to be influential for controlling the vesicle size in high transition temperature lipids, the factor FRR was found the most influential factor controlling the size of vesicles consisting of low transition temperature lipids and polymer-based nanoparticles. The biological activity of the resulting constructs was confirmed by an invitro transfection of pDNA constructs using cationic nanoprecipitated vesicles. Design of experiments and multivariate data analysis revealed the mathematical relationship and significance of the factors TFR and FRR in the microfluidics process to the liposome size, polydispersity and transfection efficiency. Multivariate tools were used to cluster and predict specific in-vivo immune responses dependent on key liposome adjuvant characteristics upon delivery a tuberculosis antigen in a vaccine candidate. The addition of a low solubility model drug (propofol) in the nanoprecipitation method resulted in a significantly higher solubilisation of the drug within the liposomal bilayer, compared to the control method. The microfluidics method underwent scale-up work by increasing the channel diameter and parallelisation of the mixers in a planar way, resulting in an overall 40-fold increase in throughput. Furthermore, microfluidic tools were developed based on a microfluidics-directed tangential flow filtration, which allowed for a continuous manufacturing, purification and concentration of liposomal drug products.

Ensemble fuzzy belief intrusion detection design

With the rapid growth of the Internet, computer attacks are increasing at a fast pace and can easily cause millions of dollar in damage to an organization. Detecting these attacks is an important issue of computer security. There are many types of attacks and they fall into four main categories, Denial of Service (DoS) attacks, Probe, User to Root (U2R) attacks, and Remote to Local (R2L) attacks. Within these categories, DoS and Probe attacks continuously show up with greater frequency in a short period of time when they attack systems. They are different from the normal traffic data and can be easily separated from normal activities. On the contrary, U2R and R2L attacks are embedded in the data portions of the packets and normally involve only a single connection. It becomes difficult to achieve satisfactory detection accuracy for detecting these two attacks. Therefore, we focus on studying the ambiguity problem between normal activities and U2R/R2L attacks. The goal is to build a detection system that can accurately and quickly detect these two attacks. In this dissertation, we design a two-phase intrusion detection approach. In the first phase, a correlation-based feature selection algorithm is proposed to advance the speed of detection. Features with poor prediction ability for the signatures of attacks and features inter-correlated with one or more other features are considered redundant. Such features are removed and only indispensable information about the original feature space remains. In the second phase, we develop an ensemble intrusion detection system to achieve accurate detection performance. The proposed method includes multiple feature selecting intrusion detectors and a data mining intrusion detector. The former ones consist of a set of detectors, and each of them uses a fuzzy clustering technique and belief theory to solve the ambiguity problem. The latter one applies data mining technique to automatically extract computer users’ normal behavior from training network traffic data. The final decision is a combination of the outputs of feature selecting and data mining detectors. The experimental results indicate that our ensemble approach not only significantly reduces the detection time but also effectively detect U2R and R2L attacks that contain degrees of ambiguous information.

On the Development of an Automated Design Procedure to Design Optimal Robots

The objective in this work is to build a rapid and automated numerical design method that makes optimal design of robots possible. In this work, two classes of optimal robot design problems were specifically addressed: (1) When the objective is to optimize a pre-designed robot, and (2) when the goal is to design an optimal robot from scratch. In the first case, to reach the optimum design some of the critical dimensions or specific measures to optimize (design parameters) are varied within an established range. Then the stress is calculated as a function of the design parameter(s), the design parameter(s) that optimizes a pre-determined performance index provides the optimum design. In the second case, this work focuses on the development of an automated procedure for the optimal design of robotic systems. For this purpose, Pro/Engineer© and MatLab© software packages are integrated to draw the robot parts, optimize them, and then re-draw the optimal system parts.

Development of a Lab-on-a-Chip Device for Rapid Nanotoxicity Assessment In Vitro

Increasing useof nanomaterials in consumer products and biomedical applications creates the possibilities of intentional/unintentional exposure to humans and the environment. Beyond the physiological limit, the nanomaterialexposure to humans can induce toxicity. It is difficult to define toxicity of nanoparticles on humans as it varies by nanomaterialcomposition, size, surface properties and the target organ/cell line. Traditional tests for nanomaterialtoxicity assessment are mostly based on bulk-colorimetric assays. In many studies, nanomaterials have found to interfere with assay-dye to produce false results and usually require several hours or days to collect results. Therefore, there is a clear need for alternative tools that can provide accurate, rapid, and sensitive measure of initial nanomaterialscreening. Recent advancement in single cell studies has suggested discovering cell properties not found earlier in traditional bulk assays. A complex phenomenon, like nanotoxicity, may become clearer when studied at the single cell level, including with small colonies of cells. Advances in lab-on-a-chip techniques have played a significant role in drug discoveries and biosensor applications, however, rarely explored for nanomaterialtoxicity assessment. We presented such cell-integrated chip-based approach that provided quantitative and rapid response of cellhealth, through electrochemical measurements. Moreover, the novel design of the device presented in this study was capable of capturing and analyzing the cells at a single cell and small cell-population level. We examined the change in exocytosis (i.e. neurotransmitterrelease) properties of a single PC12 cell, when exposed to CuOand TiO2 nanoparticles. We found both nanomaterials to interfere with the cell exocytosis function. We also studied the whole-cell response of a single-cell and a small cell-population simultaneously in real-time for the first time. The presented study can be a reference to the future research in the direction of nanotoxicity assessment to develop miniature, simple, and cost-effective tool for fast, quantitative measurements at high throughput level. The designed lab-on-a-chip device and measurement techniques utilized in the present work can be applied for the assessment of othernanoparticles' toxicity, as well.

Ensemble Fuzzy Belief Intrusion Detection Design

With the rapid growth of the Internet, computer attacks are increasing at a fast pace and can easily cause millions of dollar in damage to an organization. Detecting these attacks is an important issue of computer security. There are many types of attacks and they fall into four main categories, Denial of Service (DoS) attacks, Probe, User to Root (U2R) attacks, and Remote to Local (R2L) attacks. Within these categories, DoS and Probe attacks continuously show up with greater frequency in a short period of time when they attack systems. They are different from the normal traffic data and can be easily separated from normal activities. On the contrary, U2R and R2L attacks are embedded in the data portions of the packets and normally involve only a single connection. It becomes difficult to achieve satisfactory detection accuracy for detecting these two attacks. Therefore, we focus on studying the ambiguity problem between normal activities and U2R/R2L attacks. The goal is to build a detection system that can accurately and quickly detect these two attacks. In this dissertation, we design a two-phase intrusion detection approach. In the first phase, a correlation-based feature selection algorithm is proposed to advance the speed of detection. Features with poor prediction ability for the signatures of attacks and features inter-correlated with one or more other features are considered redundant. Such features are removed and only indispensable information about the original feature space remains. In the second phase, we develop an ensemble intrusion detection system to achieve accurate detection performance. The proposed method includes multiple feature selecting intrusion detectors and a data mining intrusion detector. The former ones consist of a set of detectors, and each of them uses a fuzzy clustering technique and belief theory to solve the ambiguity problem. The latter one applies data mining technique to automatically extract computer users’ normal behavior from training network traffic data. The final decision is a combination of the outputs of feature selecting and data mining detectors. The experimental results indicate that our ensemble approach not only significantly reduces the detection time but also effectively detect U2R and R2L attacks that contain degrees of ambiguous information.

Design construction and testing of a heat loop for study of fouling and its interrelationship to corrosion in heat exchanger tubes

A heat loop suitable for the study of thermal fouling and its relationship to corrosion processes was designed, constructed and tested. The design adopted was an improvement over those used by such investigators as Hopkins and the Heat Transfer Research Institute in that very low levels of fouling could be detected accurately, the heat transfer surface could be readily removed for examination and the chemistry of the environment could be carefully monitored and controlled. In addition, an indirect method of electrical heating of the heat transfer surface was employed to eliminate magnetic and electric effects which result when direct resistance heating is employed to a test section. The testing of the loop was done using a 316 stainless steel test section and a suspension of ferric oxide and water in an attempt to duplicate the results obtained by Hopkins. Two types of thermal ·fouling resistance versus time curves were obtained . (i) Asymptotic type fouling curve, similar to the fouling behaviour described by Kern and Seaton and other investigators, was the most frequent type of fouling curve obtained. Thermal fouling occurred at a steadily decreasing rate before reaching a final asymptotic value. (ii) If an asymptotically fouled tube was cooled with rapid cir- ·culation for periods up to eight hours at zero heat flux, and heating restarted, fouling recommenced at a high linear rate. The fouling results obtained were observed to be similar and 1n agreement with the fouling behaviour reported previously by Hopkins and it was possible to duplicate quite closely the previous results . This supports the contention of Hopkins that the fouling results obtained were due to a crevice corrosion process and not an artifact of that heat loop which might have caused electrical and magnetic effects influencing the fouling. The effects of Reynolds number and heat flux on the asymptotic fouling resistance have been determined. A single experiment to study the effect of oxygen concentration has been carried out. The ferric oxide concentration for most of the fouling trials was standardized at 2400 ppM and the range of Reynolds number and heat flux for the study was 11000-29500 and 89-121 KW/M², respectively.

An Audit and Feedback Intervention for Reducing Antibiotic Prescribing in General Dental Practice : The RAPiD Cluster Randomised Controlled Trial

Funding: This study was conducted as part of the TRiaDS programme of implementation research which is funded by NHS Education for Scotland (NES). The Health Services Research Unit which is funded by the Chief Scientist Office of the Scottish Government Health and Social Care Directorates supported the study. The funder had no influence over the design, conduct, analysis and write up of the study.

Design, Fabrication, and Characterization of Carbon Nanotube Field Emission Devices for Advanced Applications

Carbon nanotubes (CNTs) have recently emerged as promising candidates for electron field emission (FE) cathodes in integrated FE devices. These nanostructured carbon materials possess exceptional properties and their synthesis can be thoroughly controlled. Their integration into advanced electronic devices, including not only FE cathodes, but sensors, energy storage devices, and circuit components, has seen rapid growth in recent years. The results of the studies presented here demonstrate that the CNT field emitter is an excellent candidate for next generation vacuum microelectronics and related electron emission devices in several advanced applications.

The work presented in this study addresses determining factors that currently confine the performance and application of CNT-FE devices. Characterization studies and improvements to the FE properties of CNTs, along with Micro-Electro-Mechanical Systems (MEMS) design and fabrication, were utilized in achieving these goals. Important performance limiting parameters, including emitter lifetime and failure from poor substrate adhesion, are examined. The compatibility and integration of CNT emitters with the governing MEMS substrate (i.e., polycrystalline silicon), and its impact on these performance limiting parameters, are reported. CNT growth mechanisms and kinetics were investigated and compared to silicon (100) to improve the design of CNT emitter integrated MEMS based electronic devices, specifically in vacuum microelectronic device (VMD) applications.

Improved growth allowed for design and development of novel cold-cathode FE devices utilizing CNT field emitters. A chemical ionization (CI) source based on a CNT-FE electron source was developed and evaluated in a commercial desktop mass spectrometer for explosives trace detection. This work demonstrated the first reported use of a CNT-based ion source capable of collecting CI mass spectra. The CNT-FE source demonstrated low power requirements, pulsing capabilities, and average lifetimes of over 320 hours when operated in constant emission mode under elevated pressures, without sacrificing performance. Additionally, a novel packaged ion source for miniature mass spectrometer applications using CNT emitters, a MEMS based Nier-type geometry, and a Low Temperature Cofired Ceramic (LTCC) 3D scaffold with integrated ion optics were developed and characterized. While previous research has shown other devices capable of collecting ion currents on chip, this LTCC packaged MEMS micro-ion source demonstrated improvements in energy and angular dispersion as well as the ability to direct the ions out of the packaged source and towards a mass analyzer. Simulations and experimental design, fabrication, and characterization were used to make these improvements.

Finally, novel CNT-FE devices were developed to investigate their potential to perform as active circuit elements in VMD circuits. Difficulty integrating devices at micron-scales has hindered the use of vacuum electronic devices in integrated circuits, despite the unique advantages they offer in select applications. Using a combination of particle trajectory simulation and experimental characterization, device performance in an integrated platform was investigated. Solutions to the difficulties in operating multiple devices in close proximity and enhancing electron transmission (i.e., reducing grid loss) are explored in detail. A systematic and iterative process was used to develop isolation structures that reduced crosstalk between neighboring devices from 15% on average, to nearly zero. Innovative geometries and a new operational mode reduced grid loss by nearly threefold, thereby improving transmission of the emitted cathode current to the anode from 25% in initial designs to 70% on average. These performance enhancements are important enablers for larger scale integration and for the realization of complex vacuum microelectronic circuits.

Design, fabrication and characterization of resonant waveguide grating based optical biosensors

The absence of rapid, low cost and highly sensitive biodetection platform has hindered the implementation of next generation cheap and early stage clinical or home based point-of-care diagnostics. Label-free optical biosensing with high sensitivity, throughput, compactness, and low cost, plays an important role to resolve these diagnostic challenges and pushes the detection limit down to single molecule. Optical nanostructures, specifically the resonant waveguide grating (RWG) and nano-ribbon cavity based biodetection are promising in this context. The main element of this dissertation is design, fabrication and characterization of RWG sensors for different spectral regions (e.g. visible, near infrared) for use in label-free optical biosensing and also to explore different RWG parameters to maximize sensitivity and increase detection accuracy. Design and fabrication of the waveguide embedded resonant nano-cavity are also studied. Multi-parametric analyses were done using customized optical simulator to understand the operational principle of these sensors and more important the relationship between the physical design parameters and sensor sensitivities. Silicon nitride (SixNy) is a useful waveguide material because of its wide transparency across the whole infrared, visible and part of UV spectrum, and comparatively higher refractive index than glass substrate. SixNy based RWGs on glass substrate are designed and fabricated applying both electron beam lithography and low cost nano-imprint lithography techniques. A Chromium hard mask aided nano-fabrication technique is developed for making very high aspect ratio optical nano-structure on glass substrate. An aspect ratio of 10 for very narrow (~60 nm wide) grating lines is achieved which is the highest presented so far. The fabricated RWG sensors are characterized for both bulk (183.3 nm/RIU) and surface sensitivity (0.21nm/nm-layer), and then used for successful detection of Immunoglobulin-G (IgG) antibodies and antigen (~1μg/ml) both in buffer and serum. Widely used optical biosensors like surface plasmon resonance and optical microcavities are limited in the separation of bulk response from the surface binding events which is crucial for ultralow biosensing application with thermal or other perturbations. A RWG based dual resonance approach is proposed and verified by controlled experiments for separating the response of bulk and surface sensitivity. The dual resonance approach gives sensitivity ratio of 9.4 whereas the competitive polarization based approach can offer only 2.5. The improved performance of the dual resonance approach would help reducing probability of false reading in precise bio-assay experiments where thermal variations are probable like portable diagnostics.