Label-free detection of human prostate-specific antigen (hPSA) using film bulk acoustic resonators (FBARs)

Label-free detection of cancer biomarkers using low cost biosensors has promising applications in clinical diagnostics. In this work, ZnO-based thin film bulk acoustic wave resonators (FBARs) with resonant frequency of ∼1.5 GHz and mass sensitivity of 0.015 mg/m2 (1.5 ng/cm2) have been fabricated for their deployment as biosensors. Mouse monoclonal antibody, anti-human prostate-specific antigen (Anti-hPSA) has been used to bind human prostate-specific antigen (hPSA), a model cancer used in this study. Ellipsometry was used to characterize and optimise the antibody adsorption and antigen binding on gold surface. It was found that the best amount of antibody at the gold surface for effective antigen binding is around 1 mg/m2, above or below which resulted in the reduced antigen binding due to either the limited binding sites (below 1 mg/m2) or increased steric effect (above 1 mg/m2). The FBAR data were in good agreement with the data obtained from ellipsometry. Antigen binding experiments using FBAR sensors demonstrated that FBARs have the capability to precisely detect antigen binding, thereby making FBARs an attractive low cost alternative to existing cancer diagnostic sensors.This work was supported by the Engineering and Physical Sciences Research Council [grants EP/F062966/1, EP/F063865/1 and EP/F06294X/1], the Royal Society [grant RG120061] and the National Natural Science Foundation of China (NSFC) [grant 61150110485].This is the accepted manuscript version. The final published version of the article is available from Elsevier at http://www.sciencedirect.com/science/article/pii/S0925400513011052

Bulk and Surface Acoustic Wave Sensor Arrays for Multi-Analyte Detection: A Review

Bulk acoustic wave (BAW) and surface acoustic wave (SAW) sensor devices have successfully been used in a wide variety of gas sensing, liquid sensing, and biosensing applications. Devices include BAW sensors using thickness shear modes and SAW sensors using Rayleigh waves or horizontally polarized shear waves (HPSWs). Analyte specificity and selectivity of the sensors are determined by the sensor coatings. If a group of analytes is to be detected or if only selective coatings (i.e., coatings responding to more than one analyte) are available, the use of multi-sensor arrays is advantageous, as the evaluation of the resulting signal patterns allows qualitative and quantitative characterization of the sample. Virtual sensor arrays utilize only one sensor but combine itwith enhanced signal evaluation methods or preceding sample separation, which results in similar results as obtained with multi-sensor arrays. Both array types have shown to be promising with regard to system integration and low costs. This review discusses principles and design considerations for acoustic multi-sensor and virtual sensor arrays and outlines the use of these arrays in multi-analyte detection applications, focusing mainly on developments of the past decade

Love Wave Biosensors: A Review

In the fields of analytical and physical chemistry, medical diagnostics and biotechnology there is an increasing demand of highly selective and sensitive analytical techniques which, optimally, allow an in real-time label-free monitoring with easy to use, reliable, miniaturized and low cost devices. Biosensors meet many of the above features which have led them to gain a place in the analytical bench top as alternative or complementary methods for routine classical analysis. Different sensing technologies are being used for biosensors. Categorized by the transducer mechanism, optical and acoustic wave sensing technologies have emerged as very promising biosensors technologies. Optical sensing represents the most often technology currently used in biosensors applications. Among others, Surface Plasmon Resonance (SPR) is probably one of the better known label-free optical techniques, being the main shortcoming of this method its high cost. Acoustic wave devices represent a cost-effective alternative to these advanced optical approaches [1], since they combine their direct detection, simplicity in handling, real-time monitoring, good sensitivity and selectivity capabilities with a more reduced cost. The main challenges of the acoustic techniques remain on the improvement of the sensitivity with the objective to reduce the limit of detection (LOD), multi-analysis and multi-analyte detection (High-Throughput Screening systems-HTS), and integration capabilities. Acoustic sensing has taken advantage of the progress made in the last decades in piezoelectric resonators for radio-frequency (rf) telecommunication technologies. The so-called gravimetric technique [2], which is based on the change in the resonance frequency experimented by the resonator due to a mass attached on the sensor surface, has opened a great deal of applications in bio-chemical sensing in both gas and liquid media. Traditionally, the most commonly used acoustic wave biosensors were based on QCM devices. This was primarily due to the fact that the QCM has been studied in detail for over 50 years and has become a mature, commercially available, robust and affordable technology [3, 4]. LW acoustic sensors have attracted a great deal of attention in the scientific community during the last two decades, due to its reported high sensitivity in liquid media compared to traditional QCM-based sensors. Nevertheless, there are still some issues to be further understood, clarified and/or improved about this technology; mostly for biosensor applications. LW devices are able to operate at higher frequencies than traditional QCMs [5]; typical operation frequencies are between 80-300 MHz. Higher frequencies lead, in principle, to higher sensitivity because the acoustic wave penetration depth into the adjacent media is reduced [6]. However, the increase in the operation frequency also results in an increased noise level, thus restricting the LOD. The LOD determines the minimum surface mass that can be detected. In this sense, the optimization of the read out and characterization system for these high frequency devices is a key aspect for improving the LOD [7]. Another important aspect of LW technology is the optimization of the fluidics, specially the flow cell. This is of extreme importance for reducing the noise and increasing the biosensor system stability; aspects that will contribute to improve the LOD. The analysis and interpretation of the results obtained with LW biosensors must be deeper understood, since the acoustic signal presents a mixed contribution of changes in the mass and the viscoelasticity of the adsorbed layers due to interactions of the biomolecules. A better understanding of the transduction mechanism in LW sensors is a first step to advance in this issue; however its inherent complexity leads, in many cases, to frustration [8]. The fabrication process of the transducer, unlike in traditional QCM sensors, is another aspect under investigation in LW technology, where features such as: substrate materials, sizes, structures and packaging must be still optimized. This chapter aims to provide an updated insight in the mentioned topics focused on biosensors applications

Nanomaterial-Enhanced Receptor Technology for Silicon On-Chip Biosensing Application

Nanomaterials integration in biosensors designs are known to enhance sensing and signaling capabilities by exhibiting remarkably high surface area enhancement and intrinsic reactivity owing to their distinctive optical, chemical, electrical and catalytic properties. We present the synthesis and characterization of silver nanoparticles (AgNPs), and their immobilization on a silicon on-chip biosensor platform to enhance sensing capability for prostate specific antigen (PSA) - cancer biomarkers. Several techniques, including UV-Visible (UV-Vis) absorption spectrum, Fourier transforms infrared spectroscopy (FTIR), high resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM) and field emission scanning electron microscopy (FESEM) were used for characterizing the AgNPs. The biochemical sensor consists of AgNPs immobilized on the receptor layer of a silicon avalanche mode light emitting device (Si AM LED) which enables on-chip optical detection biological analytes. A bio-interaction layer etched from the chip interacts with the evanescent field of a micro dimensioned waveguide. An array of detectors below the receptor cavity selectively monitor reflected light in the UV, visible, infrared and far infrared wavelength regions. AgNPs used as an immobilization layer in the receptor layer enhances selective absorption analytes, causing a change in detection signal as a function of propagation wavelength as light is dispersed. The analytes could range from gases to cancer biomarkers like prostate specific antigen

Towards Bio-impedance Based Labs: A Review

In this article, some of the main contributions to BI (Bio-Impedance) parameter-based systems for medical, biological and industrial fields, oriented to develop micro laboratory systems are summarized. These small systems are enabled by the development of new measurement techniques and systems (labs), based on the impedance as biomarker. The electrical properties of the life mater allow the straightforward, low cost and usually non-invasive measurement methods to define its status or value, with the possibility to know its time evolution. This work proposes a review of bio-impedance based methods being employed to develop new LoC (Lab-on-a-Chips) systems, and some open problems identified as main research challenges, such as, the accuracy limits of measurements techniques, the role of the microelectrode-biological impedance modeling in measurements and system portability specifications demanded for many applications.Spanish founded Project: TEC 2013-46242-C3-1-P: Integrated Microsystem for Cell Culture AssaysFEDE

On-chip optical sensors

Adding more functionality to chips is an important trend in the advancement of technology. During the past couple of decades, integrated circuit developments have focused on keeping Moore\u27s Law alive More of Moore . Moore\u27s law predicts the doubling of the number of transistors on an integrated circuit every year. My research objectives revolve around More than Moore , where different functionalities are sought to be integrated on chip. Sensing in particular is becoming of paramount importance in a variety of applications. Booming healthcare costs can be reduced with early diagnosis, which requires improved sensitivity and lower cost. To halt global warming, environmental monitoring requires miniature gas sensors that are cheap enough to be deployed at mass scale. First, we explore a novel silicon waveguide platform that is expected to perform well as a sensor in comparison to the conventional 220 nm thick waveguide. 50 and 70 nm shallow silicon waveguides have the advantage of easier lithography than conventional 220 nm thick waveguides due to the large minimum feature size required of 1 µm. 1 µm wide waveguides in these shallow platforms are single mode. A multi-mode interference device is designed in this platform to function as the smallest MMI sensor, giving sensitivity of 427 nm / refractive index unit (RIU) at a length of 4 mm. The silicon photonic MMI sensor is based on detecting refractive index changes. Refractometric techniques such as the MMI sensor require surface functionalization to achieve selectivity or specificity. Spectroscopic methods, usually reserved for material characterization in a research setting, can be adapted for highly specific label-free sensing. Chapter 4 explores the use of a highly doped III-V semiconductor for on chip infrared spectroscopy. Finite element method and finite different time domain were both used to design a plasmonic slot waveguide for gas sensing. On chip lasers and detectors have been designed using InAs. While InAs is still considered more expensive than silicon, the electronics industry expects to start incorporating more materials in standard fabrication processes, including III-V semiconductors for their superior properties including mobility. Thus, experimental realization of this sensor is feasible. A drawback with infrared spectroscopy is that it is difficult to use with biological fluids. Chapter 5 explores the use of Raman spectroscopy as a sensing method. To adapt Raman spectroscopy for sensing, the most important task is to enhance the Raman signal. The way the Raman signal is generated means that the number of photons is generally very low and usually bulk material or concentrated fluids are used as samples. To measure low concentrations of a probe molecule, the probe molecule is placed on a surface enhanced Raman spectroscopy (SERS) substrate. A typical SERS substrate is composed of metal nanostructures for their surface plasmon resonance property, which causes a large amplification in the electric field in particular hot spots. By decorated silicon nanowires with silver nanoparticles, an enhancement factor of 1011 was realized and picomolar concentrations of pyridine were detected using Raman spectroscopy. In conclusion, this thesis provides new concepts and foundations in three directions that are all important for on chip optical sensing. First, silicon photonics is the technology of choice that is nearest to the market and a multi-mode interference sensor based on shallow silicon waveguides was designed. Further work can explore how to cascade such MMIs to increase sensitivity without sacrificing the free spectral range. Second, infrared plasmonics is a promising technology. Before semiconductor plasmonics, on chip devices operated in the visible or near IR and then microwave region of the electromagnetic spectrum. By using highly doped semiconductors, it is possible to bridge the gap and operate with mid-infrared wavelengths. The implications are highlighted by designing a waveguide platform that can be used for next generation on chip infrared spectroscopy. Third, Raman spectroscopy was exploited as a sensing technique by experimental realization of a SERS substrate using equipment-free fabrication methods

Progress in Developing a Clinically Viable Diagnostics Using Whispering Gallery Mode Resonators

Whispering gallery mode (WGM) resonators enable the label-free detection of analytes based on refractive index sensing. These WGM microsphere resonators achieve high sensitivities by confining light within their cavity. This enhances the pathlength amplifying sample-light interactions leading to improved sensing metrics. By taking advantage of their small footprint, high sensitivity and flexible design, we have developed a variety of unique label-free analytical platforms. We recently demonstrated a large scale multiplexed imaging platform where hundreds of resonators are simultaneously characterized by coupling a fluorescent dye to the resonator surface. This scheme was used to quantify several biomarkers of ovarian cancer with detection limits comparable to ELISA. Recently, we extended this technique by developing an evanescent scattering approach for characterizing the WGM resonances. Since signal levels scale with excitation power, measurements can be done with high temporal resolution using less expensive imaging equipment. This approach, therefore, offers promise for developing fast, inexpensive, sensitive detection for the multiplexed detection of disease biomarkers. Making improvements to clinical tools used for diagnosing cancer have been of considerable interest as it can drastically improve the prognosis of patients. One such example is the introduction of prostate specific antigen as an early screening tool using ELISA. Since its introduction prostate cancer mortality rates have dramatically decreased due to the detection of the disease in early stages where the cancer is more treatable. The early detection approach can be adopted for all diseases such as ovarian cancer and multiple myeloma. However, challenges in identifying a single biomarker that exists in lower, undetectable concentrations have limited the development of early screening tools for many types of cancers. Additionally, immunoassay platforms amenable to routine screening must be quick, cost-effective and easily-integrated into the clinic. WGM platforms provide an opportunity to detect and identify protein and non-protein disease biomarkers as a sensitive and multiplex approach. Additionally, WGM can be integrated with numerous fluidic platforms that improve the practical aspects of the development of a device which is critical for the translation of the tool into a clinical setting. Here, the integration of WGM detectors into platforms such as capillary electrophoresis (CE) and droplet analysis will be presented. Separation of serum using CE presents the advantage of separation non-diagnostically relevant proteins which can reduce non-specific binding. The progress of developing a WGM resonators platform for the diagnostics of multiple myeloma using CE-WGM separations will be discussed

Ultrafast Microfluidic Immunoassays Towards Real-time Intervention of Cytokine Storms

Biomarker-guided precision medicine holds great promise to provide personalized therapy with a good understanding of the molecular or cellular data of an individual patient. However, implementing this approach in critical care uniquely faces enormous challenges as it requires obtaining “real-time” data with high sensitivity, reliability, and multiplex capacity near the patient’s bedside in the quickly evolving illness. Current immunodiagnostic platforms generally compromise assay sensitivity and specificity for speed or face significantly increased complexity and cost for highly multiplexed detection with low sample volume. This thesis introduces two novel ultrafast immunoassay platforms: one is a machine learning-based digital molecular counting assay, and the other is a label-free nano-plasmonic sensor integrated with an electrokinetic mixer. Both of them incorporate microfluidic approaches to pave the way for near-real-time interventions of cytokine storms. In the first part of the thesis, we present an innovative concept and the theoretical study that enables ultrafast measurement of multiple protein biomarkers (<1 min assay incubation) with comparable sensitivity to the gold standard ELISA method. The approach, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA) incorporates the single-molecular counting of proteins at the early, pre-equilibrium state to achieve the combination of high speed and sensitivity. We experimentally demonstrated the assay’s application in near-real-time monitoring of patients receiving chimeric antigen receptor (CAR) T-cell therapy and for longitudinal serum cytokine measurements in a mouse sepsis model. In the second part, we report the further development of a machine learning-based PEdELISA microarray data analysis approach with a significantly extended multiplex capacity using the spatial-spectral microfluidic encoding technique. This unique approach, together with a convolutional neural network-based image analysis algorithm, remarkably reduced errors faced by the highly multiplexed digital immunoassay at low analyte concentrations. As a result, we demonstrated the longitudinal data collection of 14 serum cytokines in human patients receiving CAR-T cell therapy at concentrations < 10pg/mL with a sample volume < 10 µL and 5-min assay incubation. In the third part, we demonstrate the clinical application of a machine learning-based digital protein microarray platform for rapid multiplex quantification of cytokines from critically ill COVID-19 patients admitted to the intensive care unit. The platform comprises two low-cost modules: (i) a semi-automated fluidic dispensing module that can be operated inside a biosafety cabinet to minimize the exposure of technician to the virus infection and (ii) a compact fluorescence optical scanner for the potential near-bedside readout. The automated system has achieved high interassay precision (~10% CV) with high sensitivity (<0.4pg/mL). Our data revealed large subject-to-subject variability in patient responses to anti-inflammatory treatment for COVID-19, reaffirming the need for a personalized strategy guided by rapid cytokine assays. Lastly, an AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biosensing device was presented for rapid analysis of cytokine IL-1β among sepsis patients. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The technologies developed have enabled new capabilities with broad application to advance precision medicine of life-threatening acute illnesses in critical care, which potentially will allow the clinical team to make individualized treatment decisions based on a set of time-resolved biomarker signatures.PHDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163129/1/yujing_1.pd

Microfluidics for Biosensing and Diagnostics

Efforts to miniaturize sensing and diagnostic devices and to integrate multiple functions into one device have caused massive growth in the field of microfluidics and this integration is now recognized as an important feature of most new diagnostic approaches. These approaches have and continue to change the field of biosensing and diagnostics. In this Special Issue, we present a small collection of works describing microfluidics with applications in biosensing and diagnostics

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