Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed

Study of Hydrogel Properties and Immobilization of a Bioluminescent Bioreporter

The past decade has witnessed the development of a novel class of sensors; Bioluminescent Bioreporter Integrated Chip biosensors, designed to accurately measure small physical changes in the atmosphere using genetically engineered micro-organisms (bioreporters). The major challenge that now remains is to design a suitable matrix that can hold the bioreporters functionally active over a period of time. The project is an effort to develop and demonstrate alternative methods to favorably immobilize bioreporters without affecting its metabolic functions. The wide collection of literature indicates the successful use of hydrogels for cellular immobilization over the past few years. Hydrogels are inexpensive, easy to fabricate in laboratory conditions, chemically inert, biocompatible, structurally stable, optically transparent and more importantly permeable to target analytes. The equilibrium swelling properties and membrane potential of the hydrogels were studied to gain sufficient insight into its characteristic response over long periods. The knowledge was then used to control the mechanical properties such as stiffness, porosity and surface charge of the hydrogel scaffolds to exactly meet the design criteria of an ideal immobilization matrix. The study particularly involved tests to immobilize cells of Pseudomonas fluorescens 5RL, a genetically engineered bioluminescent bioreporter in the volume and surface of charged polyelectrolyte hydrogels and alginate gels. The bioluminescent light assays, Live dead assays, Electron microscopy were used to verify the viability of the immobilized bioreporters. The tests demonstrate the ability of the hydrogels in immobilizing the microorganisms without significantly affecting the physiology of the cells. The results indicate tremendous potential and a major role that hydrogels can play in the immobilization of microorganisms. Such successful techniques integrated with large scale commercialization could change the face of the conventional sensor technology in every possible areas like waste water remediation, medical diagnostics, sealed room gas analyzers in space shuttles and many more

Towards improved biomonitoring tools for an intensified sustainable multi-use environment.

The increasing use of our environment for multiple contrasting activities (e.g. fisheries, tourism) will have to be accompanied by improved monitoring of environmental quality, to avoid transboundary conflicts and ensure long-term sustainable intensified usage. Biomonitoring approaches are appropriate for this, since they can integrate biological effects of environmental exposure rather than measure individual compound concentrations. Recent advances in biomonitoring concepts and tools focus on single-cell assays and purified biological components that can be miniaturized and integrated in automated systems. Despite these advances, we are still very far from being able to deploy bioassays routinely in environmental monitoring, mostly because of lack of experience in interpreting responses and insufficient robustness of the biosensors for their environmental application. Further future challenges include broadening the spectrum of detectable compounds by biosensors, accelerate response times and combining sample pretreatment strategies with bioassays

A CMOS microluminometer for use in a bioluminescent bioreporter integrated circuit

This thesis presents the analysis, design, fabrication, and testing of a second generation microluminometer for use in an electronic/biological chemical sensor known as a Bioluminescent Bioreporter Integrated Circuit (BBIC). The microluminometer consists of photodetection (light to analog) and signal processing (analog to digital) all integrated in a standard HP 0.5 µm CMOS process. The basic operation of photodetection is first explained in detail and the available CMOS photodetectors are presented. A system-level overview of the microluminometer components is explored and error sources from each element are introduced. The focus of this thesis is on the redesign of the switched capacitor integrator within the microluminometer. Problems from the first design are illustrated and areas for improvements are identified. The implementation and design of these improvements are described in detail and simulations are made to verify the design. Finally, the results from testing of the fabricated prototype are presented and significant improvements compared to the first generation prototype are illustrated

A Low Power CMOS Microluminometer and Transmitter for Bioluminescent Bioreporter Integrated Circuit (BBIC)

This thesis is a study of the design of a low power CMOS microluminometer and transmitter for bioluminescent bioreporter integrated circuit (BBIC). A BBIC sensor chip with lower consumption was fabricated in the 0.35μm CMOS process. This design was an improvement over a previous BBIC [1]. The previous BBIC was designed using a different CMOS process (0.5μm) and a different CAD tool (Magic). This thesis work involves redesign of the chip in 0.35μm CMOS process using Cadence design tool with improvement for power dissipation. Larger resisters are used instead of several small resisters, which were placed between power supply and ground and consumed too much power in the previous chip [1]. Also, the bias currents for several amplifiers were reduced to decrease the power consumption even further. The chip was tested under normal light condition and it was verified that the device implemented the basic functions of a sensor. The power consumption has been reduced to 3.5% of the previous chip [1], which is not because of the feature size change. Some test results of the photodiode and signal processing circuit are given. The transmitter system was designed using CAD tools Cadence following previous work [2]. The power amplifier was added to the transmitter to give larger signal out of the circuit. The simulation was run in Cadence. Appendixes show all the net list files for the sensor chip and transmitter circuit

Low power design of a 916 MHz Gilbert Cell Mixer and a Class-A Power Amplifier for Bioluminescent Bioreporter Integrated Circuit Transmitter

This thesis presents the low power design of a 916MHz Gilbert cell mixer and a Class-A power amplifier for the Bioluminescent Bioreporter Integrated Circuit (BBIC) transmitter. There has been increased use in the man-made sensors which can operate in environments unsuitable for humans and at locations remote from the observer. One such sensor is the bioluminescent bioreporter integrated circuit (BBIC). Bioluminescent bioreporters are the bacteria that are genetically engineered in order to achieve bioluminescence when in contact with the target substance. The BBIC has bioreporters placed on a single CMOS integrated circuit (IC) that detects the bioluminescence, performs the signal processing and finally transmits the senor data. The wireless transmission allows for remote sensing by eliminating the need of costly cabling to communicate with the sensor. The wireless data transmission is performed by the transmitter system. The digital data stream generated by the signal processing circuitry of the BBIC is ASK modulated for transmission. The direct conversion transmitter used in this design includes a PLL, Mixer and a Power amplifier. The PLL is used to generate a 916MHz frequency signal. This signal is mixed with the digital data signal generated from the signal processing circuitry of the BBIC. A double balanced Gilbert cell is used to perform the mixing operation. The mixer output is applied to a power amplifier which provides amplification of the RF output power. The Gilbert cell mixer and the power amplifier have been implemented in 90nm CMOS process available through MOSIS

Bioluminesoivien makrolidibioreportterien optimointi

Antimikrobilääkkeiden lisääntynyt käyttö kuormittaa ympäristöä ja kiihdyttää antimikrobiresistenssin muodostumista taudinaiheuttajilla. Bakteerien lisääntynyt vastustuskyky antibiootteja kohtaan vaikuttaa sekä ihmisten että eläinten terveyteen. Ympäristön antibioottipitoisuuksia seurataan jatkuvasti erilaisin kemiallisin menetelmin, vaikka ne eivät kuvaa antimikrobilääkkeiden biosaatavuutta. Bioreportteri-bakteerit on kehitetty havaitsemaan erilaisia ympäristölle haitallisia aineita, kuten antibiootteja. Näitä eläviä, geneettisesti muunneltuja bakteereita voidaan käyttää näytteen biosaatavan osuuden määrittämisessä. Bioreportterien avulla voidaan näin ollen kartoittaa antimikrobiresistenssin leviämistä. Tämän tutkielman tavoitteena oli kehittää ja valmistaa paranneltu bioluminesenssia tuottava bioreportteri makrolidiantibioottien havaitsemiseen. Työn tarkoituksena oli yhdistää mrx geeni ja mph(A)R repressorigeeni mph(A) makrolidiresistenssioperonin promoottorialueeseen ja lusiferaasioperoni luxCDABE reportterigeeneihin. Tutkielman päätavoitteena oli kartoittaa makrolidiresistenssioperoni mph(A):n koodaaman oletetun hydrofobisen transmembraani- ja solukuljetusproteiini Mrx:n vaikutus bioreportterin herkkyyteen ja induktioaikaan. Tavoitteena oli myös optimoida kolmen aiemmin kehitetyn bioreportterin käyttöä muiden makrolidien kuin erytromysiinin kanssa. Tutkielmassa mrx-mph(A)R-geenifragmentti kloonattiin osaksi pmph(A)luxCDABE-vektoria, jonka jälkeen yhdistelmäplasmidi transformoitiin Escherichia coli DH10B-kantaan. Oikeanlaisen rakenteen todentamisen jälkeen pmph(A)luxCDABE-mrx-mph(A)R-plasmidin toimivuutta verrattiin kolmeen jo aiemmin kehitettyyn bioreportteriin erilaisissa erytromysiini-, tylosiini- ja klindamysiinipitoisuuksissa. Uuden bioluminesenssia tuottavan makrolidibioreportterin kloonaus suoritettiin työssä onnistuneesti. Bioreportteri ei kuitenkaan reagoinut erytromysiinin, tylosiinin tai klindamysiinin kanssa lusiferaasi-entsyymiä tuottaen. Valoreaktio oli havaittavissa aiemmin kehitetyillä bioreporttereilla melko korkeissa antibioottipitoisuuksissa, mutta erytromysiinin lisäksi myös tylosiinilla ja klindamysiinilla. Lopputuloksena voidaan todeta, että mrx geenin ja sen koodaaman proteiinin kartoittamista tulisi syventää. Tämän tutkimuksen tulosten perusteella jo kehitettyjä makrolidibioreporttereita voidaan hyödyntää myös muiden makrolidien kuin erytromysiinin ja linkosamidien havaitsemiseen.The increasing use of antimicrobials causes a heavy pollution load on the environment and can enhance antimicrobial resistance of pathogenic bacteria, thus having a negative impact on human and animal health. Antibiotic concentrations in the environment are constantly monitored, however traditional chemical analyses fail to provide data on the bioavailability of antimicrobials. Whole-cell bacterial bioreporters have been developed to detect a wide variety of environmental pollutants including antimicrobials. These living, genetically engineered organisms can also be used for the measurement of the bioavailable fraction in a sample and thus bioreporters could give insights on the role of antimicrobial pollution in the dissemination of antimicrobial resistance. The aim of this study was to design and construct an improved bioluminescent bioreporter for detection of macrolide antibiotics. The mrx gene and the mph(A)R repressor gene were coupled with the mph(A) promotor of the macrolide resistance operon mph(A) and the reporter genes of the luciferase operon luxCDABE. The main objective was to determine whether Mrx, the hydrophobic and putative transmembrane transport protein of the macrolide resistance operon mph(A), would improve the sensitivity and reduce the induction time. Another aim was to optimize the use of three existing bioreporters with other macrolides than erythromycin, which was used earlier in testing their performance. The mrx-mph(A)R fragment was cloned into the pmph(A)luxCDABE vector, and the bioreporter plasmid was introduced to Escherichia coli strain DH10B. After verification of the construct pmph(A)luxCDABE-mrx-mph(A)R, the usability of the new whole-cell biosensor was compared against the three existing macrolide bioreporters with three different macrolide and lincosamide antibiotics, erythromycin, tylosin and clindamycin. The cloning of the new bioluminescent bioreporter for macrolides was performed successfully. However, the addition of erythromycin, tylosin or clindamycin to a suspension of E. coli DH10B(pmph(A)luxCDABE-mrx-mph(A)R) did not stimulate the expression of the lux genes. High concentrations of all three antibiotics triggered a light response with the existing bioreporters although the response was slow. The results indicated that further studies on the mrx gene and its encoded Mrx protein are still needed. The response of the mph(A) operon to other macrolide and lincosamide antibiotics than erythromycin was a positive and encouraging finding, since this enables detection of other synthetic macrolides than erythromycin as well as lincosamides with the existing bioreporters after optimization

A low-noise microluminometer for a bioluminescent bioreporter integrated circuit

This thesis presents the analysis and design of a low-noise microluminometer for a hybrid electronic/biological chemical sensor known as a Bioluminescent Bioreporter Integrated Circuit (BBIC). The microluminometer consists of photodetection and signal processing Both functions are integrated in a standard bulk CMOS process (HP 0.5 urn CMOS). The photodetection is first described in terms of physical operation. The implementation of photodetectors in a CMOS integrated circuit process is then presented. The signal processing system is analyzed, and the errors introduced by individual system components are described. A detailed system-level noise analysis is also presented The design of a low-noise amplifier is the focus of this thesis. The amplifier design is described in detail. Finally, the results from testing of the fabricated prototype are presented

Exploiting bioluminescence to enhance the analytical performance of whole-cell and cell-free biosensors for environmental and point-of-care applications

The routine health monitoring of living organisms and environment has become one of the major concerns of public interest. Therefore, there has been an increasing demand for fast and easy to perform monitoring technologies. The current available analytical techniques generally offer accurate and precise results; however, they often require clean samples and sophisticated equipment. Thus, they are not suitable for on site, real-time, cost-effective routine monitoring. To this end, biosensors represent suitable analytical alternative tools. Biosensors are analytical devices integrating a biological recognition element (i.e. antibody, receptor, cell) and a transducer able to convert the biological response into an easily measurable analytical signal. These tools can easily quantify an analyte or a class of analytes of interest even in a complex matrix, like clinical or environmental samples, thanks to the specificity of the biological components. Whole-cell biosensors among others offer unique features such as low cost of production and provide comprehensive functional information (i.e. detection of unclassified compounds and synergistic effects, information about the bioavailable concentration). During this PhD, several bioengineered whole-cell biosensors have been developed and optimized for environmental and point-of-care applications. Analytical performance of biosensors have been improved (i.e. low limit of detection, faster response time and wider dynamic range) thanks to synthetic biology and genetic engineering tools. Bacterial, yeast and 3D cell cultures of mammalian cell lines have been tailored at the molecular level to improve robustness and predictivity. Several reporter genes, i.e. colorimetric, fluorescent and bioluminescent proteins, have been also profiled for finding the best candidate for each point-of-need application. Furthermore, spectral resolution of different optical reporter proteins has been exploited and multiplex detection has been achieved. The inclusion of viability control strains provided a suitable tool for assessing non-specific effects on cell viability, correcting the analytical signal and increasing the analytical performance of ready-to-use cartridges