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In this thesis, three different read-out techniques for biomolecular recognition have been studied. All three techniques rely on the change in dynamic behaviour of probe functionalised magnetic beads after binding to a biomolecular target complementary to the probe. In the first technique presented, the sample is exposed to an AC magnetic field while the response to this field is probed using a laser source and a photodetector positioned at opposite sides of the sample. Beads bound to the target entity will experience an increase in their hydrodynamic volume, and will not be able to respond as rapidly to an alternating field as free beads. Here, the target entity is either DNA coils formed by rolling circle amplification or biotinylated bovine serum albumin (bBSA). The change in dynamic behaviour is measured as a frequency dependent modulation of transmitted light. Limit of detections (LODs) of 5 pM DNA coils originating from a V. cholerae target and 100 pM of bBSA have been achieved. In the second technique presented, the beads are magnetically transported across a probe functionalised detection area on a microchip. Beads bound to a target will be blocked from interaction with the detection area probes, whereas in the absence of a target, beads will be immobilised on the detection area. The LOD of biotin for this system proved to be in the range of 20 to 50 ng/ml. In the third technique presented, the sample is microfluidically transported to a detection area on a microchip. The read-out is performed using a planar Hall effect bridge sensor. A sinusoidal current is applied to the bridge in one direction and the sensor output voltage is measured across the sensor in the perpendicular direction. The AC current induced bead magnetisation contributing to the sensor output will appear different for free beads compared to beads bound to a target. LODs of 500 B. globigii spores and 2 pM of V. cholerae DNA coils were achieved. From a lab-on-a-chip point of view, all three techniques considered in this thesis show promising results with regards to sensitivity and integrability.

Although DNA microarray can detect multiple DNA samples simultaneously, current detection techniques involve PCR and other traditional procedures. In this study, a sensitive, specific and rapid detection method, which eliminates PCR and other lengthy processes, for pathogenic DNA is presented. This technology is based on the hybridization of target DNA to the immobilized probe, extension of probe DNAs using the target-DNA as a template and signal generation by streptavidin-horseradish peroxidase and substrate. This method is highly specific and sensitive, allowing single-nucleotide-base mismatches discrimination and the detection at femtomole level. The experiments are designed to achieve short hybridization time. Therefore, satisfactory signal can be detected within minutes, allowing the rapid detection of multiple pathogenic DNA. Most importantly, the E. coli genomic DNA can be detected using this technology. In conclusion, this detection method is useful for applications including on-site pathogenic disease detection, crime scene investigation, and pathogen inspection in the environment.

The detection of particular genetic sequences aids in the early detection and diagnosis of disease; permits monitoring of the health and state of the natural environment; and informs forensic investigations. To date, gene detection has relied on enzymatic amplification followed by optical readout. Though these technologies have advanced dramatically, the instruments and assays are costly and lack portability. The work presented herein addresses an urgent challenge: molecular diagnostics at the point-of-need. This work reports the first electronic chip capable of analyzing - directly, without amplification, and with clinically-relevant sensitivity - multiple genes of interest present in a clinical sample. It reports a dramatic acceleration in sample-to-answer times, with clinically actionable findings in minutes where legacy techniques take hours or days. The key to the sensitivity and speed of the biosensors reported herein lies in their architecture and morphology on multiple lengthscales. It is proven that hybridization-based assays employing a nucleic probe attached to a solid surface can only achieve efficient performance when displayed on a nanotextured surface. It is also discovered that these same sensing elements must reach tens of micrometers into solution to achieve rapid, sensitive detection of nucleic acids in clinical samples. As a result, the materials integrated onto the sensing chip reported herein are engineered on multiple lengthscales - from the nanometers to the tens of micrometers. Engineering is done through a combination of low-cost, convenient top-down photolithographic patterning; combined with hierarchically-designed bottom-up growth of electrodeposited sensing elements. The capstone of this work is a chip that distinguishes among different types of bacteria in an unpurified sample. The chip gives accurate answers in under half an hour when detecting bacteria at a level of 1.5 colony-forming-unit (cfu) per microliter. These speeds and sensitivies enable the application of this technology in point-of-need assays for infectious disease detection. Ultimately, the work showcases the power of bringing together techniques and principles from materials chemistry, biochemistry, applied physics, and electrical engineering to the solution of an important problem relevant to human health.

The detection of particular genetic sequences aids in the early detection and diagnosis of disease; permits monitoring of the health and state of the natural environment; and informs forensic investigations. To date, gene detection has relied on enzymatic amplification followed by optical readout. Though these technologies have advanced dramatically, the instruments and assays are costly and lack portability. The work presented herein addresses an urgent challenge: molecular diagnostics at the point-of-need. This work reports the first electronic chip capable of analyzing - directly, without amplification, and with clinically-relevant sensitivity - multiple genes of interest present in a clinical sample. It reports a dramatic acceleration in sample-to-answer times, with clinically actionable findings in minutes where legacy techniques take hours or days. The key to the sensitivity and speed of the biosensors reported herein lies in their architecture and morphology on multiple lengthscales. It is proven that hybridization-based assays employing a nucleic probe attached to a solid surface can only achieve efficient performance when displayed on a nanotextured surface. It is also discovered that these same sensing elements must reach tens of micrometers into solution to achieve rapid, sensitive detection of nucleic acids in clinical samples. As a result, the materials integrated onto the sensing chip reported herein are engineered on multiple lengthscales - from the nanometers to the tens of micrometers. Engineering is done through a combination of low-cost, convenient top-down photolithographic patterning; combined with hierarchically-designed bottom-up growth of electrodeposited sensing elements. The capstone of this work is a chip that distinguishes among different types of bacteria in an unpurified sample. The chip gives accurate answers in under half an hour when detecting bacteria at a level of 1.5 colony-forming-unit (cfu) per microliter. These speeds and sensitivies enable the application of this technology in point-of-need assays for infectious disease detection. Ultimately, the work showcases the power of bringing together techniques and principles from materials chemistry, biochemistry, applied physics, and electrical engineering to the solution of an important problem relevant to human health.

Recently, photoelectrochemical (PEC) signal transduction, with optical excitation and electronic readout, has been identified as a powerful transduction strategy for bioanalysis due to its high sensitivity and low limit-of-detection. Semiconductive materials have been used as the building blocks of PEC transducers, while plasmonic nanoparticles (NPs) are frequently used as signal amplifiers in these biosensors. Though these approaches have been previously used in PEC biosensing, the interaction between plasmonic and semiconductors NPs linked together through biomolecules are not currently well-understood. Herein, we developed new strategies for preparing photoelectrodes using solution-based methods to enhance the photocurrent of PEC transducers. These transducers were then used to investigate the interaction mechanisms between plasmonic NPs and the photoelectrodes with the goal of enhancing the limit-of-detection of PEC biosensors. In order to create photoelectrodes that were fabricated using facile benchtop methods designed to enhance the photocurrent of PEC transducers, wrinkled scaffolds were used to fabricate photoelectrodes that show an order of magnitude enhancement in photocurrent compared to the planar electrodes. These electrodes were further used in label-free signal-off DNA biosensing without any amplification steps. Limit-of-detection of 200 times lower were reported using these wrinkled photoelectrodes, than planar electrodes. Gold (Au) and TiO2¬ NPs were used as model materials to investigate the interaction between plasmonic and semiconductor NPs on a photoelectrode. The modulation of photocurrent was examined by varying the concentration of Au NPs and under different optical excitation wavelengths. UV light excitation provided larger photocurrent enhancement – at low concentration of Au NP – than visible light excitation. Furthermore, anodic photocurrent generation efficiencies by the photoelectrodes, which were prepared by using only Au NPs, were compared between interband and intraband excitation. The Au NP photoelectrodes demonstrated higher anodic photocurrent at interband excitation than intraband excitation and were further optimized by varying the size and deposition time of the Au NPs. Following this, Au NP- labeled DNA was used to study the effect of the distance between Au NPs and TiO2 NPs on the magnitude of the measured photocurrent. When Au NPs were in proximity with TiO2, they increased the generated photocurrent; however, they reduced the measured photocurrent when they were positioned further away from TiO2 NPs. Utilizing this switching behavior of PEC signals, a differential signal generation strategy was adopted to achieve a biosensor with enhanced sensitivity and signal-to-noise ratio. Ultimately, we designed a PEC signal transduction strategy to detect nucleic acids without target labeling. In this assay, Au NP-labeled DNA was used as a signal-amplification-barcode that was introduced to the assay following target binding. This label-free PEC biosensor showed a low limit-of-detection (3 fM), broad (1 fM – 100 pM) linear range, and capability to detect single and double base-mismatched sequences of DNA. Thus, this work presents materials and signal transduction innovations that enhance the performance metrics of biosensors. / Dissertation / Doctor of Philosophy (PhD) / Detection and quantification of biomolecules is of utmost importance in early diagnosis, disease monitoring, prognosis, and disease management. In the past few decades, enormous efforts have been put towards utilizing photoelectrochemical (PEC) processes for biomolecular detection due to their high sensitivity. Gold nanoparticles are frequently being used to amplify the signal in the PEC bio-detection assay due to their plasmonic properties. However, the exact nature of the interaction between gold nanoparticles and the electrode material has not been determined. In this thesis, we investigated the interaction of gold nanoparticles with photoelectrode materials when they are separated by nucleic-acid sequences. Excitation energy and nucleic-acid length were varied to modulate the PEC current. The improved understanding of this interaction was further utilized to achieve a programmable response of nucleotide sensor from the photoelectrodes upon detecting the analyte of interest. We further developed different types of biosensing assay designs and examined their performance in terms of limit-of-detection, sensitivity, and specificity. Finally, we developed a new class of biosensor for detecting nucleic acids in bodily fluid and assessed the assay by using both electrochemical and PEC signal readout.

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The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to the detection probe ZFP, which allows for generating chemiluminescent signal in streptavidin-HRP (Horseradish peroxidase) assay upon ZFPs binding to their target DNA. Our assay generates DNA-dependent signal and allows for a detection limit of 0.5 nM without DNA amplification or DNA labeling. Our system can be developed into a simple multiplexed detection diagnostic for multiplexed detection of dsDNA.

This thesis describes a new approach to biomolecular analysis, called the volume-amplified magnetic nanobead detection assay (VAM-DNA). It is a sensitive, specific magnetic bioassay that offers a potential platform for the development of low-cost, easy-to-use diagnostic devices. The VAM-NDA consists of three basic steps: biomolecular target recognition, enzymatic amplification of the probe-target complex using the rolling circle amplification (RCA) technique, and addition of target complementary probe-tagged magnetic nanobeads which exhibit Brownian relaxation behavior. Target detection is demonstrated by measuring the frequency-dependent complex magnetization of the magnetic beads. The binding of the RCA products (target DNA-sequence coils) to the bead surface causes a dramatic increase in the bead size, corresponding essentially to the size of the DNA coil (typically around one micrometer). This causes a decrease in the Brownian relaxation frequency, since it is inversely proportional to the hydrodynamic size of the beads. The concentration of the DNA coils is monitored by measuring the decrease in amplitude of the Brownian relaxation peaks of free beads. The parameters oligonucleotide surface coverage, bead concentration, bead size and RCA times were investigated in this thesis to characterize features of the assay. It was found that all of these parameters affect the outcome and efficiency of the assay. The possibility of implementing the assay on a portable, highly sensitive AC susceptometer platform was also investigated. The performance of the assay under these circumstances was compared with that using a superconducting quantum interference device (SQUID); the sensitivity of the assay was similar for both platforms. It is concluded that, the VAM-NDA opens up the possibility to perform biomolecular detection in point-of-care and outpatient settings on portable platforms similar to the one tested in this thesis. Finally, the VAM-NDA was used to detect Escherichia coli bacteria and the spores of Bacillus globigii, the non-pathogenic simulant of Bacillus anthracis. A limit of detection of at least 50 bacteria or spores was achieved. This shows that the assay has great potential for sensitive detection of biomolecules in both environmental and biomedical applications.