Thin Film Based Biosensors for Point of Care Diagnosis of Cortisol

This dissertation explores the different ways to create thin film-based biosensors that are capable of rapid and label-free detection of cortisol, a non-specific biomarker closely linked to stress, within the physiological range of 10pM to 10 uM. Increased cortisol levels have been linked to stress-related diseases, such as chronic fatigue syndrome, irritable bowel syndrome, and post-traumatic stress disorder. It also plays a role in the suppression of the immune system as well. Therefore, accurate measurement of cortisol in saliva, serum, plasma, urine, sweat, and hair, is clinically significance to predict physical and mental diseases. In this dissertation, thin film-based electrochemical immunosensors were fabricated using a self-assembled monolayer (SAM) functionalized by cortisol specific antibodies to detect cortisol at 10 pM level sensitivities in the presence of a redox probe. The fabricated electrochemical cortisol immunosensors were able to detect cortisol in human saliva samples and the outcomes were validated using the standard Enzyme Linked Immuno Sorbent Assay (ELISA) technique. With the aim of improving signal amplification and label-free cortisol detection, copper nanoparticles were incorporated on screen-printed carbon electrodes (SPCE) for the fabrication of electrochemical cortisol immunosensor. This SPCE-based sensor showed a sensitivity of 4.21µA/M and the limit of detection 6.6nM. Both the SAM and SPCE-based immunosensors were not thermally stable due to the instability of antibodies at room temperature. To address this issue, an antibody-free immunosensor was fabricated. Molecular Imprinted Polymer (MIP) was used to template the target cortisol molecule. The MIP-based sensing platform was prepared using polypyrrole, a thermally stable conducting polymer. The conductivity of the polymer ensured good electrical performance. The polypyrrole-based MIP was synthesized by means of electrochemical polymerization and was used to detect cortisol within the physiological range at room temperature. MIP-based sensors exhibited the detection limit of 1 pM, and were cost-effective, easy to fabricate, temperature stable, and reusable. The sensing performance of the resulting sensors was comparable to those of commercially available technologies, such as ELISA. Aiming to perform cortisol sensing at point-of-care (POC), an Extended Gate Field Effect Transistor (EGFET) was integrated with a developed MIP cortisol sensor. The as developed MIP-EGFET sensor was used to detect the cortisol concentration in the range of 1 pM to 100 nM. A few of the major advantages of the developed sensor are its ability to provide a direct readout and simpler electronic systems, which are necessary for miniaturized Point of Care devices

Ion-Sensitive Field-Effect Transistor for Biological Sensing

In recent years there has been great progress in applying FET-type biosensors for highly sensitive biological detection. Among them, the ISFET (ion-sensitive field-effect transistor) is one of the most intriguing approaches in electrical biosensing technology. Here, we review some of the main advances in this field over the past few years, explore its application prospects, and discuss the main issues, approaches, and challenges, with the aim of stimulating a broader interest in developing ISFET-based biosensors and extending their applications for reliable and sensitive analysis of various biomolecules such as DNA, proteins, enzymes, and cells

Surface composition of mixed self-assembled monolayers on Au by infrared attenuated total reflection spectroscopy

Abstract Self-assembled monolayers (SAMs) of N-(2-hydroxyethyl)-3-mercaptopropanamide (NMPA) were synthesized directly on the surface of electron-beam evaporated Au films, starting from 3-mercaptopropionic acid (3MPA) via ethyl-3-(3-dimethylamino-propyl)carbodiimide/N-hydroxysulfosuccinimide sodium salt (EDC/NHSS) coupling with ethanolamine hydrochloride. The influence on the reaction yield of the acidity of EDC/NHSS solutions (pH = 5.6 or 4.8) was assessed by exploiting the high surface sensitivity of infrared attenuated total reflection spectroscopy. The light-matter interaction was modeled in the framework of a matrix formalism considering the complete multi-layer sample structure. A comparison between the relative intensity of the main absorption bands, associated with amide I and carbonyl stretching of carboxylic acid or amide II vibrations, with a calibration curve obtained from the measurement of mixed 3MPA/NMPA SAMs, show that the more acid solution is 16% more efficient. This is mostly due to the higher protonation of the 3MPA

Real-Time, Selective Detection of Heavy Metal Ions in Water Using 2d Nanomaterials-based Field-effect Transistors

Excessive intake of heavy metals damages the central nervous system and causes brain and blood disorders in mammals. Heavy metal contamination is commonly associated with exposure to mercury, lead, arsenic, and cadmium (arsenic is a metalloid, but classified as a heavy metal). Traditional methods to detect heavy metal ions include graphite furnace atomic absorption spectroscopy (GFAAS), inductively-coupled plasma optical emission spectroscopy (ICP-OES), and inductively-coupled plasma mass spectroscopy (ICP-MS). Recently, many new methods have been proposed to detect heavy metal ions, including atomic absorption spectrometry, fluorescent sensors, colorimetric sensors, electrochemical sensors, X-ray absorption fine structure spectroscopy, ultrasensitive dynamic light scatting assays, and ion selective electrodes. Although significant progress has been made, there are still some critical issues to be addressed, e.g., lack of portability, the need for well-trained personnel, highly expensive and complex instruments, long response time (tens of minutes or even longer), and the possibility of introducing additional contamination. Therefore, it is highly desirable to develop a real-time, low-cost, portable, user-friendly analytical platform for rapid inline analysis of mercury, lead and other heavy metal ions. This dissertation research aims to investigate field-effect transistor (FET) sensors based on two-dimensional (2D) nanomaterials with specific probe-functionalized gold (Au) nanoparticles (NPs). The fundamental mechanism of the FET platform is to use a 2D nanomaterial as the conducting channel to transport charge carriers (electrons or holes). Upon the capture of target analytes, the charge carrier concentration and/or mobility changes correspondingly with a signal of current change within the channel. As a result, the FET characteristic changes upon the introduction of the heavy metal ion solution, varies with the metal concentration, and takes only a few seconds to respond. Control experiments are performed to verify the selectivity of the 2D nanomaterial/Au NP hybrid sensor to specific targets. The rapid, selective, sensitive, and stable detection performance indicates the promise of 2D nanomaterial/Au NP hybrid sensors for heavy metal ion detection in an aqueous solution. This research is accomplished through several steps: First, various heavy metal ion contaminants, their damage, and the conventional detection methods are reviewed; Second, the FET-based plaform and its working mechanism are explored; Third, the understanding of various 2D nanomaterials, their unique properties pertinent to electronic sensing, and their selection to realize real-time, selective, and sensitive detection of heavy metal ions is carried out; Finally, improvement of stability, sensitivity and lifetime of FET sensors is investigated. In this thesis work, sensitive and selective FET-based 2D nanomaterial/Au NP hybrid sensors for Pb2+, Hg2+, As(III), and As(V) have been demonstrated. The 2D nanomaterials include reduced graphene oxide (rGO), molybdenum disulfide (MoS2), and black phosphorus (BP). The hybrid structure consists of a nanomaterial film, homogeneously dispersed Au NPs, and specific probes. The detection is enabled by recording the electrical conductance of the device through monitoring the change in the drain current of the 2D nanomaterial sheets. The platform offers a promising route for real-time (1-2 seconds), high-performance and low-cost detection of heavy metal ions. The lower detection limit can reach the order of µg/L (parts-per-billion or ppb). The sensor also shows high selectivity against other co-existing metal ions. To improve the sensitivity of the nanomaterial-based electronic sensor, theoretical analysis on the sensing mechanism has been carried out, together with experimental validation. Theoretical analysis indicates that sensitivity-related factors are semiconducting properties of nanomaterials (e.g., carrier mobility, band gap), number of probes, and adsorption capacity of Au NPs. Experimental results suggest that a higher sensitivity for sensors can be realized by forming hybrid structures with thinner 2D conducting materials with a larger band gap and a higher carrier mobility, increasing the areal density of anchoring sites on the sensor surface, and enhancing the adsorption of detection probes. Investigation into the stability of the nanomaterial-based electronic sensor includes the binding strength between the nanomaterial and electrodes, stability of the nanomaterials in ambient environment and water, the detachment of Au NPs, the lifetime and diffusion of probes, and the overall stability of the sensor platform. Subsequently, strategies to improve the stability of the nanomaterial-based FET sensor have been proposed. Finally, the FET sensor has been used for the accurate prediction of arsenic ions in lake water and integrated into a practical flowing water system for continuous detection of lead ions. The rapid, selective, sensitive, and stable detection performance of the FET sensor for various heavy metal ions in water suggests a promising future for in-situ detection of contamination events. The thesis study provides a scientific foundation to engineer FET sensors with enhanced performance. An attempt has been made to practically develop the FET platform into standalone sensors and to integrate the sensor into flowing water equipment for heavy metal ion detection. The thesis results thus contribute to the future application of FET sensors for monitoring water contamination and mitigating the public health risk

Micro- and nano-devices for electrochemical sensing

Electrode miniaturization has profoundly revolutionized the field of electrochemical sensing, opening up unprecedented opportunities for probing biological events with a high spatial and temporal resolution, integrating electrochemical systems with microfluidics, and designing arrays for multiplexed sensing. Several technological issues posed by the desire for downsizing have been addressed so far, leading to micrometric and nanometric sensing systems with different degrees of maturity. However, there is still an endless margin for researchers to improve current strategies and cope with demanding sensing fields, such as lab-on-a-chip devices and multi-array sensors, brain chemistry, and cell monitoring. In this review, we present current trends in the design of micro-/nano-electrochemical sensors and cutting-edge applications reported in the last 10 years. Micro- and nanosensors are divided into four categories depending on the transduction mechanism, e.g., amperometric, impedimetric, potentiometric, and transistor-based, to best guide the reader through the different detection strategies and highlight major advancements as well as still unaddressed demands in electrochemical sensing

Charge-Modulated Extended Gate Organic Field Effect Transistor for Biosensing Applications

The interest in organic field effect transistors (OFETs) employed as a biosensing platform has grown in recent years, driven largely by the potential to create inexpensive, sensitive analytical devices with a wide range of chemical and biological sensing applications. A particularly promising architecture for these type of devices is the Charge-Modulated Organic Field-Effect Transistor (CM-OFET). In the CM-OFET, a control gate electrode is capacitively coupled to a floating gate and used to bias the OFET, eliminating the need for an additional, often macroscale, reference electrode. In addition, charge accumulated in a designated sensing region of the floating gate modulates the output source drain current, ISD, of the transistor, providing sensing activity that is spatially separated from the organic semiconductor layer. Here, a CM-OFET based on solution processed Tips-pentacene as the organic semi-conductor that is both low cost and very simple to fabricate is reported. The CM-OFET biosensors fabricated here were predominantly based on the widely used Si/SiO2 substrates, where the degenerately doped Si acted as the gate electrode with a SiO2 dielectric layer. A limited number of Al/Al2O3 based CM-FETS are also presented. This thesis includes a detailed description of fabrication of these CM-OFET devices alongside a detailed discussion of the principle of operation, both as organic transistors and as analytical for monitoring pH and protein detection. The thesis focusses primarily on the characteristics of CM-OFET devices based on the Si/SiO2 substrate. The fabrication of Si/SiO2 CM-OFETs was very simple, requiring only a single lithography or shadow evaporation stage. Despite the simplicity, the CM-OFETs reliably displayed electrical characteristics typical of organic field effect transistors. The electrical characteristics were reproducible with over 90% yield. However, the responses of the devices when tested for pH sensing and protein detection, were inconsistent and with large error. Further analysis of the CM-OFET architecture revealed limitations associated with the geometrical layout of the Si/SiO2 CM-OFET device may have caused this deficiency in sensing response. A modified CM-OFET employing Al/Al2O3 as gate and gate dielectric layers was designed in which the geometry was optimized to maximise sensitivity to changes in charge within the sensing region. A process for the fabrication of the Al/Al2O3 CM-OFET was developed and the Al-based CM-OFETs were found to exhibit behaviour typical of an organic transistor, albeit with relatively lower source drain current compared to Si/SiO2 CM-OFET devices. Due to limited time, the sensitivity of the Al-based CM-OFET was not fully characterized. Further work regarding the enhancement of the device’s charge carrier mobility of the device and particularly, experimental investigation of the Al/Al2O3 CM-OFET for sensing applications is needed

Design of Surface Modifications for Nanoscale Sensor Applications

Nanoscale biosensors provide the possibility to miniaturize optic, acoustic and electric sensors to the dimensions of biomolecules. This enables approaching single-molecule detection and new sensing modalities that probe molecular conformation. Nanoscale sensors are predominantly surface-based and label-free to exploit inherent advantages of physical phenomena allowing high sensitivity without distortive labeling. There are three main criteria to be optimized in the design of surface-based and label-free biosensors: (i) the biomolecules of interest must bind with high affinity and selectively to the sensitive area; (ii) the biomolecules must be efficiently transported from the bulk solution to the sensor; and (iii) the transducer concept must be sufficiently sensitive to detect low coverage of captured biomolecules within reasonable time scales. The majority of literature on nanoscale biosensors deals with the third criterion while implicitly assuming that solutions developed for macroscale biosensors to the first two, equally important, criteria are applicable also to nanoscale sensors. We focus on providing an introduction to and perspectives on the advanced concepts for surface functionalization of biosensors with nanosized sensor elements that have been developed over the past decades (criterion (iii)). We review in detail how patterning of molecular films designed to control interactions of biomolecules with nanoscale biosensor surfaces creates new possibilities as well as new challenges

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

Early diagnosis in a premalignant (or pre-invasive) state represents the only chance for cure in neoplastic diseases such as pancreatic-biliary cancer, which are otherwise detected at later stages and can only be treated using palliative approaches, with no hope for a cure. Screening methods for the purpose of secondary prevention are not yet available for these cancers. Current diagnostic methods mostly rely on imaging techniques and conventional cytopathology, but they do not display adequate sensitivity to allow valid early diagnosis. Next-generation sequencing can be used to detect DNA markers down to the physical limit; however, this assay requires labeling and is time-consuming. The additional determination of a protein marker that is a predictor of aggressive behavior is a promising innovative approach, which holds the potential to improve diagnostic accuracy. Moreover, the possibility to detect biomarkers in blood serum offers the advantage of a noninvasive diagnosis. In this study, both the DNA and protein markers of pancreatic mucinous cysts were analyzed in human blood serum down to the single-molecule limit using the SiMoT (single-molecule assay with a large transistor) platform. The SiMoT device proposed herein, which exploits an inkjet-printed organic semiconductor on plastic foil, comprises an innovative 3D-printed sensing gate module, consisting of a truncated cone that protrudes from a plastic substrate and is compatible with standard ELISA wells. This 3D gate concept adds tremendous control over the biosensing system stability, along with minimal consumption of the capturing molecules and body fluid samples. The 3D sensing gate modules were extensively characterized from both a material and electrical perspective, successfully proving their suitability as detection interfaces for biosensing applications. KRAS and MUC1 target molecules were successfully analyzed in diluted human blood serum with the 3D sensing gate functionalized with b-KRAS and anti-MUC1, achieving a limit of detection of 10 zM and 40 zM, respectively. These limits of detection correspond to (1 ± 1) KRAS and (2 ± 1) MUC1 molecules in the 100 μL serum sample volume. This study provides a promising application of the 3D SiMoT platform, potentially facilitating the timely, noninvasive, and reliable identification of pancreatic cancer precursor cysts

Design and optimization of ultrathin silicon field effect transistor's for sensitive, electronic-based detection of biological analytes

Noncommunicable diseases (NCD) are currently the leading cause of death worldwide. Over 57 million deaths occur globally each year, with close to 36 million of them attributed to NCD’s, and 80% of those in low and middle income countries. Most of these were due to such chronic illnesses as cancer, cardiovascular disease, diabetes, and lung disease. Moreover, the prevalence of these diseases is rising fastest in low-income regions which have little resources to combat these large, yet avoidable costs. In particular, over 1.6 million cases of cancer are caused each year in the United States, with nearly 600,000 of these cases being fatal. Cancer is an uncontrolled growth and spread of abnormal cells in the body, and unfortunately, can exist in many different cell types. The complexity in the causes of cancer has made it tougher to diagnose since several factors may weight into its prevalence such as: genetic factors, lifestyle factors, certain types of infections, and different environmental exposures. As a result, the protocols for the most cost-effective intervention are available across four main approaches to cancer prevention and control: primary prevention, early detection, treatment, and palliative care. Early diagnosis based on awareness of early signs and symptoms and, if affordable, population-based screening improves survival, particularly for breast, cervical, colorectal, skin and oral cancers. If primary prevention of cancer fails, secondary prevention (early detection) may be the difference between irreversible spread of a malignant cancer, and the patient’s survival. Early detection commonly refers to the diagnosis of a disease before individuals show obvious signs or symptoms of illness. With cancer, RNA and protein biomarkers of cells are currently assayed to determine their serums level and if they have deviated from the normal ranges. However, these assays commonly require large centralized lab facilities, frequent monitoring during treatment, and expensive equipment and/or supplies. This has led to point-of-care diagnostics becoming a $16 billion global market, aimed at miniaturizing technology and making it cost-effective for individual patient testing and treatment without the use of centralized lab facilities. A main point-of-care testing platform being pursued utilizes Complementary Metal Oxide Semiconductor (CMOS) technology. CMOS-based products can enable clinical tests to be conducted in a fast, simple, safe, and reliable manner, with improved sensitivities. Moreover, CMOS products offer portability and low power consumption, in large part due to the explosion in the semiconductor and communications markets. Silicon nanowires are of great interest for point-of-care testing as they are a CMOS compatible structure, require the use of no labels, and are highly sensitive to the binding of molecules to their surfaces. This is due to the large surface area to volume ratio afforded to nanowires. Moreover, arrays of silicon nanowires have demonstrated multiplexed, label-free sensing of cancer markers from undiluted serum samples. However, the research going into CMOS for point-of-care is in its infancy compared to other optical (surface plasmon resonance, fluorescence) or electrochemical methods (glucose sensors), although the technology for CMOS has been around for decades. Thus, the protocols for optimization of the sensors and their bioconjugation have not matured to the point DNA microarrays and ELISA’s have. The protocols for creation of a dependable silicon nanowire biosensor revolve around three main aspects: semiconductor processing, device functionalization, and choice of analytes. In this dissertation, I discuss our efforts to create a stable, silicon nanowire based sensor using CMOS compatible techniques and optimization processes. Moreover, I talk about our efforts into creating a device functionalization protocol using monofunctional silanes which affords the best sensitivity and specify for an electronic based biosensor. Finally, I discuss our look towards the future in silicon nanowires by using high-k dielectrics in our fabrication process, as well as an alternative monolayer deposition method which utilizes sub-nanometer thickness poly-l-lysine monolayers, for sensing clinically relevant targets of microRNA. Using a special type of silane, called a monofunctional silane, and a vapor based deposition method, we were able to achieve sub-nanometer levels functional monolayers on thermally oxidized silicon surfaces. We employed a variety of characterization techniques (XPS, AFM, ellipsometry) to determine the densities of the monolayer, uniformity, topography, and their point of saturation. Furthermore, we demonstrate this method’s applicability to biosensors by using it to functionalize substrates for silicon nanowires, gold nanoparticles, and protein microarrays. In tandem with this work, we constructed a “top down” silicon nanowire processing protocol which yielded nanowires capable of long-term, stable measurements in aqueous solutions. The combination of anneals, dry etching, and final wet etching gave mV standard deviations in device threshold characteristics. This protocol combined with the monolayer protocol above allowed an in-depth characterization of the pH sensitivity of bare devices, ones with silanes, and ones conjugated with proteins to be determined. Similarly, different oxide thicknesses and their effect on device sensitivity for proteins were also explored. Using a bunch of different linker chemistries and characterizing their conjugation of antibodies through fluorescence and the device, allowed for a chemistry to be chosen which was used to sense mouse immunoglobulins in pg/mL levels with high specificity. Finally, we take the fabrication of nanowires to the next level by using high-k dielectrics (HfO2) as the gate insulator. We deposit HfO2 through ALD (atomic layer deposition) and optimize the anneals to provide nanowires with ~200mV subthreshold slopes, sub-mV threshold deviations, and sub nanoampere gate leakages. All these characteristics exceed the processes for thermal oxide gated silicon nanowires, some by an order of magnitude. Since HfO2 is a high-k material, reaction of silanes and its density were unknown, but high-k materials do form stable amide linkages. Thus, we optimized a wet deposition of small molecular weight poly-l-lysine to provide a sub-nm conjugation layer for proteins and nucleotides by using AFM, XPS, and ellipsometry to understand the process. Using these combined protocols, we were able to conjugate probe oligonucleotides to surfaces and detect target microRNA’s down to 100fM concentrations, with a dynamic range over 4 orders of magnitude. With these ranges well within the clinical levels (1pM-100pM), we believe silicon nanowires have the capability to become a well-established point-of-care diagnostic platform