Smart acquisition system development for multiple readout from quantum dots’ fluorescence

Universidade do Minho School of Engineering Leonardo Lopes Ramalho Smart Acquisition System Development for Multiple Readout from Quantum Dots’ Fluorescence October, 2022 Universidade do Minho School of Engineering Leonardo Lopes Ramalho Smart Acquisition System Development for Multiple Readout from Quantum Dots’ Fluorescence Master Thesis Master in Physics Engineering Specialization in Devices, Microsystems and Nanotechnologies Work developed under the supervision of: Paulo Mateus Mendes October, 2022 STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the Universidade do Minho. , (Place) (Date) (Leonardo Lopes Ramalho) ii COPYRIGHT AND TERMS OF USE OF THIS WORK BY A THIRD PARTY This is academic work that can be used by third parties as long as internationally accepted rules and good practices regarding copyright and related rights are respected. Accordingly, this work may be used under the license provided below. If the user needs permission to make use of the work under conditions not provided for in the indicated licensing, they should contact the author through the RepositoriUM of Universidade do Minho. License granted to the users of this work Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International CC BY-NC-SA 4.0 https://creativecommons.org/licenses/by-nc-sa/4.0/deed.en This document was created with the (pdf/Xe/Lua)L A T EX processor and the NOVAthesis template (v6.10.10) [1]. iii Acknowledgements From the bottom of my heart, I would like to express my deepest gratitude to whoever impacted my life, directly or indirectly, through these last five years of an academic adventure and I want to specifically thank: — To my advisor, Paulo Mateus Mendes, for always showing me the light at the end of the tunnel, for the motivating words and advices, for the honesty and for never giving up on me. — To my co-workers in Sensovann, standing out, Nuno Pires, Gonçalo Almeida and Lu Yao, for giving me an amazing opportunity to work on this project and for making my work the best as possible. — To my colleagues from USN, Hedda Stadheim, Kristian Langvik, Kristian Tangen and Per Magne, for being an amazing product design team, for helping me with my project, for teaching me so much and for letting me work on your project. — To my beloved best friend, Rita Linda, for always remembering the person I am, for the experiences, for showing how life can be challenging and fun, and for making me a better person. — To my closest family, highlighting my parents, José Carlos and Carmen, for always giving me green card on everything, for the amazing childhood, for the sacrifices, for the opportunities, for the patience, for the continuous and unconditional support, for the values and for the love. — To my Epic friend, Jorge Daniel, for keeping me company every night. — To the best group of friends, my dear Babies K, for making the best get-togethers, for remembering I still make a difference three thousand kilometres away and for never putting me out of the picture. — To my electronics lab partner, Filipa Mota, for helping me pass all the subjects, for pushing me to work harder and for making these last three years easier. — To my university colleagues, from who I would like to stand out: Baptista, Bruno, Gama and Vilarinho, for making the online classes more lively, for showing me that we are all in the same boat and for remembering that life is tired. (”Que vida cansada”) iv Abstract Smart Acquisition System Development for Multiple Readout from Quantum Dots’ Fluorescence The increasing demand for rapid testing stimulates the development of new techniques in clinical diagnosis. When compared to other conventional methods, lateral flow immunoassay has many advantages such as low cost, easy operation, friendly use and quick response. However, it can only give a qualitative or semi quantitative result. Improving the sensibility of the test result and make it quantifiable is a very challenging field of research. Recently, luminescent nanoparticles have been used, and low detection limits have been obtained. One example is quantum dots, which are ideal fluorescent labels and can improve widely the detection sensitivity thanks to their narrow emission spectra, broad excitation range and excellent brightness. This work was focused on making a reader that could read strips targeting biomarkers of COPD (Chronic Obstructive Pulmonary Disease). The new reader was created without a motor dependency, which generally takes a great part of the battery power and the reader’s size. It consists of an acquisition board, a system board and an optical filter. Several simulations and practical tests were made to complete its instrumentation circuit. In order to get dynamic results, a Python Graphic User Interface was made, making it easier for the user to analyse. At last, the reader could perform a 563 ms read, sending the information wirelessly to the computer using a BLE protocol. The device has a changeable sensitivity through programming and a directly proportional response to light intensity. Keywords: acquisition system, BLE, LFIA, microcontroller, COPD, quantum dots, quantum dot’s fluorescence v Resumo Desenvolvimento de um Sistema de Aquisição Inteligente para Leitura Múltipla da Fluorescência de Pontos Quânticos A crescente necessidade para testes rápidos estimulou o desenvolvimento de novas técnicas na área clínica. Quando comparado com outros métodos convencionais, o imunoensaio de fluxo lateral possui muitas vantagens tais como baixo custo, fácil operação, uso amigável e resposta rápida. No entanto, só pode dar um resultado qualitativo ou semi quantitativo. Melhorar a sensibilidade do resultado do teste e torná-lo quantificável é uma área de investigação desafiante. Recentemente, nanopartículas luminescentes têm sido utilizadas, e foram alcançados limites mínimos de deteção. Um exemplo são pontos quânticos, que são marcadores fluorescentes ideais e podem melhorar amplamente a sensibilidade de deteção graças aos seus espectros de emissão estreitos, ampla faixa de excitação e excelente brilho. Este trabalho teve como foco fazer um leitor que pudesse ler tiras direcionadas aos biomarcadores de DPOC (Doença Pulmonar Obstrutiva Crónica). O novo leitor foi criado sem dependência motora, o que geralmente consome grande parte da energia da bateria e do tamanho do leitor. É constituído por uma placa de aquisição, uma placa de sistema e um filtro ótico. Várias simulações e testes práticos foram realizados para completar o seu circuito de instrumentação. Para obter resultados dinâmicos, foi feita uma Interface Gráfica de Utilizador em Python, facilitando a análise do utilizador. Por fim, o leitor conseguia realizar uma leitura em 563 ms, enviando as informações por ligação sem fio BLE para o computador. O dispositivo tem uma sensibilidade variável por meio de programação e uma resposta diretamente proporcional à intensidade da luz. Palavras-chave: sistema de aquisição, BLE, LFIA, microcontrolador, PDOC, quantum dots, quantum dots’ fluorescence vi Contents List of Figures ix 1 Introduction 1 1.1 Chronic Obstructive Pulmonary Disease . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Motivation and Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Contributions................................... 2 1.4 ThesisOrganization................................ 3 2 Disease Diagnostic Tests 4 2.1 Lateral Flow Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.2 Labels used in LFIA’s development . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2.1 Gold Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2.2 QuantumDots.............................. 6 2.2.3 Superparamagnetic nanoparticles . . . . . . . . . . . . . . . . . . . . . 7 2.2.4 Enzymes................................. 8 2.2.5 Liposomes................................ 8 2.3 QuantumDots .................................. 9 2.4 Reading Quantum Dots’ Fluorescence . . . . . . . . . . . . . . . . . . . . . . . 9 3 Acquisition System 11 3.1 ReaderArchitecture................................ 11 3.2 Opticalsystem .................................. 12 3.3 ProposedApproach................................ 13 3.4 Photodetector................................... 14 3.5 Conclusion.................................... 17 4 Acquisition Module 18 4.1 Microcontroller .................................. 19 vii 1.4. THESIS ORGANIZATION 1.4 Thesis Organization This document follows the following structure: • In the first chapter, a contextualization of diagnose tests and the importance of their accuracy is introduced, alongside with a short explanation of COPD and its diagnosing significance. This chapter ends with the motivation and contributions of this thesis. • The second chapter contains a review of different types of diagnostic tests, more specifically LFIA, and their possible labels for targeting. • Chapter 3 explains the work done to accomplish the acquisition system. A photodetector was selected, an instrumentation setup was created and simulated, and an electronic circuit was made. • The next chapter, chapter 4, contains the reader’s creation process, from the microcontroller’s selection to the reader’s casing and components placement. The device’s characterization is also performed to provide a complete analysis of the reader. • Lastly, the fifth chapter is constituted by the conclusion and the future work of this project. 3 2 Disease Diagnostic Tests The biggest problem of chronic diseases, such as COPD, is that they usually are not diagnosed at early stages, when it is easily treatable, but better detected when the disease has reached a more complicated stage. For this reason, low cost and quick tests are more required and there is a strong motivation to improve them, giving a quicker and more reliable result. 2.1 Lateral Flow Immunoassay Lateral flow technology has been available for a long time and these days it got more popular with SARSCoV-2 rapid tests [4]. They have attracted interest due to their friendly user formats, short assay times, little interferences, low costs, and being easily operated by non-specialized personnel. In simple words, a lateral flow immunoassay is an easy to handle diagnostic device used to confirm the presence or absence of a target analyte, such as pathogens or biomarkers in humans or animals, or contaminants in water supplies [5]. These tests usually have a control line to confirm if the test is working properly, along with one or more test lines. They can be qualitative and read easily (with an answer yes or no), or they can provide quantitative data when combined with a reader technology. Commonly, the LFIA technology comprises cellulose and nitrocellulose membranes, coloured nanoparticles, and antibodies as recognition elements. When a sample is added, it will flow laterally along the test device, passing through the conjugate pad into the nitrocellulose membrane and then onto the absorbent pad. (Figure 1) The sample pad acts as the first stage of the absorption process, and in some cases contains a filter, to ensure the accurate and controlled flow of the sample. The conjugate pad, which stores the conjugated labels and antibodies, will receive the sample. If the target is present, the immobilized conjugated antibodies and labels will bind to the target by immuneaffinity and continue to migrate along the test. As the sample moves along the device, the antibody-target complexes moving through the nitrocellulose membrane will bind to the second antibody anchored at the test line. A colored line will form, and 4 2.2. LABELS USED IN LFIA’S DEVELOPMENT Figure 1: LFIA Strip Schematic the intensity of the line color will vary depending on the quantity of the present target. Some targets may require quantification to determine target concentration. This is where a rapid test can be combined with a reader to provide quantitative results. The sample will pass through the nitrocellulose membrane into the absorbent pad. The absorbent pad will absorb the excess sample. The specification of the absorbent pad will have an impact on the volume of sample that the test can incorporate [6]. 2.2 Labels used in LFIA’s development Many LFIA tests are qualitative and semi-quantitative. A number of researchers developed quantitative LFIAs by coupling a signal transducer, such as optical, electrical or magnetic reader. Also, these transducers convert the coloured zones into digital signals. In optical detection, an array scanner is used as a transducer, and is a high-cost device, restricting the applications. The electrochemical detection is suitable for quantitative analysis, but it requires an additional dissolution step, since the usage of strong acids and toxic mercury limits the applications [7]. In colorimetric detection, the quantitative test results are based on the reading of coloured bands with a portable strip reader. The peak areas are correlated with the intensities of the band and indicate the concentration of analyte in the sample. In multiplex detection, a strip reader records multiple bands simultaneously and shows the individual peak for each band. Nanomaterials have attracted great interests because of its signal amplification property to achieve high sensitivity and selectivity for target analysis in LFIA. There are many examples of lateral flow assays that combine nanomaterials with immunochromatographic tests, and which can be used for clinical, environmental, and food analysis. Nanoparticles (gold nanoparticles, carbon nanoparticles), luminescent nanoparticles (quantum dots, fluorescent quenching material, up-converting phosphor), superparamagnetic nanoparticles, liposome and enzymes are used as a label in the LFIA development. Giving all the information, such as, production cost, sensibility, testing time, among other, the quantum dots seem to be the most promising label for a LFIA strip. 5 CHAPTER 2. DISEASE DIAGNOSTIC TESTS 2.2.1 Gold Nanoparticles Colloidal gold or gold nanoparticles are commonly used as detector reagent in the LFIA strip for visualization of signals. Vivid and strength red colour enhances visual detection. Other unique properties are the high chemical stability, large specific area, easy synthesis, low cost and easy preparation steps. These properties make the analysis time short and provide reliable analysis on-site [8–11]. The shape, size and stability of AuNPs (Gold Nanoparticles) are key parameters that affect the success of the LFIA. The size and colour of AuNPs depend on the amount of sodium citrate that is used in the reduction of gold [12]. AuNPs with the diameter smaller than 15 nm were small for generating a strong colour; however, nano-colloidal gold with a diameter of 20 nm was used as detector reagent. AuNPs with the diameter larger than 60–70 nm were easily self-aggregated [13] and unstable. Gold nanoparticle-labelled lateral flow immunoassay (AuNP-LFIA) is an effective method that is widely used in many fields. But the traditional AuNP-LFIA is limited due to its low sensitivity. New methods have been developed for improving sensitivity, such as silver deposition, and signal amplification of large AuNPs. Silver deposition requires additional pretreatment methods and signal amplification of large AuNPs method is simply amplifying the signals. 2.2.2 Quantum Dots Recently, in a number of studies, fluorescent nanoparticles are used rather than colorimetric markers and low detection limits are obtained [14]. Quantum dots are ideal fluorescent labels and have been widely used to improve the detection sensitivity of LFIA owing to their narrow emission spectra, broad excitation range and high fluorescent quantum yields. Quantum dots have excellent brightness, size-tunable fluorescence emissions, large absorption coefficients, good stability, and high signal-to-noise ratio. The excellence of brightness and exceptional stability of quantum dots enable them to be detected and be quantified sensitively. For simultaneous detection of several compounds in a sample, two different formats of LFIA have been developed. In the first format, LFIA includes different lines for each analyte, this is, the different antibodies of analytes are immobilized in different lines. In the second format, LFIA includes one line for both analytes, this is, all the antibodies are in the same line. An example of the first format was developed in [15], which is a ”traffic light” format lateral flow immunoassay and in this case, the LFIA detected three different antibiotics. Antibodies against antibiotics were conjugated with water-soluble quantum dots with emission maximum at either 525, 585 or 625 nm, making the strip with the colours green, yellow and red respectively, like a traffic light. The quantitative detection was based on the colour intensities of the corresponding test lines. In Figure 2, it is possible to see this format of multiple detection. In A, the strip is presented before performing any test. B shows the results for the sample containing the first antibiotic, this is, the green target. And in C, it is a strip used with a sample containing the second and third antibiotic, this is, the yellow and the red targets. 6 2.2. LABELS USED IN LFIA’S DEVELOPMENT Figure 2: Principle of detection of three antibiotics using ”traffic light” [15] In Figure 3 is represented a schematic illustration for the test strip for the second case. For the simultaneous detection, one test and one control line were constructed on the nitrocellulose membrane. The fluorescence intensities of tumour markers captured on the test and control lines were measured simultaneously by using a strip reader. Figure 3: Schematic illustration of simultaneous detection with only one test line [16] 2.2.3 Superparamagnetic nanoparticles Superparamagnetic nanoparticles (MNPs) are new labelling materials to develop a LFIA. They increase the sensitivity almost 10 to 100 times. Additionally, magnetic signals are produced by MNPs, and they are stable over long periods of time. They have low background noise, because the magnetic material does not exist usually in the environment or in the tested samples [17]. 7 CHAPTER 2. DISEASE DIAGNOSTIC TESTS Most studies have shown that super-paramagnetic particles are new attractive material to construct a LFIA, which will replace traditional labels due to its stable magnetic signal which can be entirely captured by devices, thus improving the sensitivity of LFIA. The reasons for using these particles as labels are their unique properties, such as magnetism in strong magnetic fields, and this specific property prevents aggregation and precipitation [17]. The small size of magnetic nanoparticles (MNPs) enables short detection time. The increased magnetite content of MNPs provides a stronger signal; also, the magnetite content has positive linear relationship with the signal intensity [18]. In [19], a LFIA using magnetic particles is presented exploiting spontaneous magnetic switching, a superparamagnetic characteristic, to produce a quantifiable electromagnetic induction in an alternating current carrier. This approach is practical as it does not require the application of external magnetic fields. 2.2.4 Enzymes In the literature, three types of test strips have been used. The most common strip is based on capturing an analyte by an antibody labelled with colloidal gold, latex bead or fluorescent dyes to form antibody-antigen complex. This forming complex binds to other immobilized antibody on TL and CL lines. The second type of strip is based on chemical reactions which gives a colour change. The third type is based on the enzyme-substrate reactions for colour development. The formed colour can be visualized by naked eye and standard colour chart. This leads to imprecision caused by large variation in judgment due to human errors. Quantifications of signal by readers have been also developed, for example the refractometer for determining the optical density, the electrochemical signal for measuring the conductivity of metal labels and stripping voltammetric detection of metal ion labels. However, the requirements of expensive reading device make the assay costly less beneficial. 2.2.5 Liposomes Liposomes are sphere-shaped artificial vesicles consisting of one or more phospholipid bilayers. Owing to their size and hydrophobic and hydrophilic character (besides biocompatibility), they have been particularly used in drug delivery. Liposomes’ properties differ considerably with lipid composition, surface charge, size, and the preparation method. They are very stable, and their large internal volume provides interaction with several biological elements, such as peptides, hormones, antibodies, sugars, and nucleic acids. These properties make them useful in field-portable or point-of-care sensor systems. But, these come with some major disadvantages such as the structural weakness toward detergents, pH changes which is an adverse effect on the ionic charges in the phospholipid bilayer, and osmotic pressure related to swelling or crenation [20]. 8 2.3. QUANTUM DOTS 2.3 Quantum Dots Quantum dots are semiconductor particles with a size of a few nanometres. The selection of quantum dot materials has primarily been driven by the ability to prepare particles with the desired optical properties [21]. These luminescent nanoparticles are really special because they are particles confined to three dimensions, which makes its density of states unique. The energy levels are very discrete and precise (Figure 4(a)) and the energy gap changes according to the quantum dot’s dimension. (Figure 4(b)) (a) Evolution of the density of states (DOS) with dimensionality of the system [22] (b) Relation between the size of QD and its band gap and color of fluorescence [23] Figure 4: Quantum Dots Properties Quantum dots also have an optical property, which is the main interest in this project. Quantum dots’ fluorescence is, in simple words, when a quantum dot is illuminated, absorbing the energy from the photons, it emits the light back in a different wavelength. This wavelength is related to its energy gap and, as mentioned above, is directly associated with its size. (Figure 4(b)) One of the most known applications are quantum dots LEDs. These can reach from 1250 to 67840 𝑐𝑑/𝑚2 of luminance [24]. Looking from the photodetector’s perspective, a group of quantum dots can be seen as a light source with isotropic properties and a coherent intensity of light, therefore, a light source such as a torch or a smartphone can be used as a replacement. 2.4 Reading Quantum Dots’ Fluorescence In order to read the fluorescence of quantum dots, a preliminary light emission is necessary to give energy to the quantum dots. In this case, the reader has an emitter to excite the quantum dot and a receiver to collect the light. Usually, the wavelengths of the excitation light and emission light are well-defined and really distinct [25]. This is a good advantage feature on behalf of the specific energy gap, which defines the wavelength of emission light and minimizes any interference between the LED and the photodetector [26]. The emission light, on a macroscopic scale, is isotropic, this is, the radiation propagates in the same intensity when measured in different directions. Although there are many advantages to acquire the light, 9 CHAPTER 2. DISEASE DIAGNOSTIC TESTS the disadvantages can’t be ignored. It is possible to implement the whole optical system (emitter and receiver) in the same plane, however, if the strip has several test lines, crosstalk may lead to interference in the light collected by photodetector, due to the present simultaneous multiple lines. For this project, the LFIA strips were already manufactured, and they use quantum dots as label for the targets. They are core/shell quantum dots made from CdSe/ZnS. The absorption wavelength is around 365 nm, this is, at UV light, and the emission peak wavelength is at 610 nm, which is the visible spectrum, the colour red. Figure 5 is a spectrum from typical quantum dots that were manufactured. Figure 5: Absorption and Emission spectrum of CdSe/ZnS quantum dots 10 3 Acquisition System An existing prototype that makes a reliable and quick result has been accomplished. However, the reader is bulky, heavy, and not user-friendly. In order to read all the strip’s T-Lines (test lines), this prototype uses a black box with the optical system, which has the emitter and the detector, and a motor to make the strip move, in other words, it is the opposite of a scanner, the sensor is fixed in the same place and the strip moves to be scanned. 3.1 Reader Architecture Below, Figure 6 represents a schematic of this working reader. Figure 6: Reader Block Diagram, showing a general overview of the device’s architecture The reader is composed by a battery and its charging port, a system board, a measuring board, the optical system (black box), a motor and a platform to place and move the strip. The system board controls the entire reader and makes the bridge between the user and the board. It has a power switch to preserve battery, two LEDs to indicate the power state and the Bluetooth connection, 11 CHAPTER 3. ACQUISITION SYSTEM a microcontroller, several surface mounted devices for the instrumentation and connectors for the other board (measuring board) and a motor. The measuring board is made only to excite and collect the fluorescence from the quantum dots. It has a UV LED with its own LED drive, so the intensity of the excitation light is always constant and the photodetector where its peak of sensibility is around the quantum dots’ emission light wavelength. 3.2 Optical system (a) Block Diagram (b) Photo Figure 7: Optical System In Figure 7(b), it is possible to see the existing prototype reader’s optical system. It is composed by a UV LED (on the left) with its own lens to focus the excitation beam. In the middle there is a dichroic mirror which reflects light below approximately 425 𝑛𝑚 and lets pass through the emission light. Figure 8 shows its transmission rate versus the wavelength. On top, there is a red filter to eliminate any light noise and increase the photodetector sensibility Figure 8: Spectrum of Dichroic Mirror [27] and a small lens to focus the light to the photodetector. At the end, there is a photodiode to convert the light into current that later will be transformed to voltage. 12 4.1. MICROCONTROLLER For the reader to work, a user and a computer are needed. Figure 16 shows a sequence diagram of the reader’s operation. Starting with the user, he scans for Bluetooth devices using the computer. After getting the list of devices, selects the desired device, which in this case is called “KOLS reader”. Then, after connecting successfully with the device, it sends the signal to start the reading. The Bluetooth module transmits this information to the microcontroller that initiates the reading by sending the signal to the photodetector. In the meantime, the photodetector is giving back the information needed, which is collected by the microcontroller’s ADC. This value is then sent to the Bluetooth module that sends wirelessly to the computer. Finally, the computer crunches the data and shows a graph, so the user can see and analyse. All the code can be consulted in Appendix A. 4.1 Microcontroller In order to control the photodetector and to make the user experience the easiest possible, a CURIOSITY HIGH PIN COUNT (HPC) DEVELOPMENT BOARD from Microchip was acquired. Figure 17: Curiosity High Pin Count Development Board This board came with a 𝑃𝐼𝐶18𝐹47𝑄10 microcontroller, which is a chip with several features, from which we can highlight: • Selectable clock up to 64 𝑀𝐻𝑧 • Three 8-bit Timers •128𝐾Bytes Program Flash Memory • Operating Voltage between 1,8𝑉and 5,5𝑉 • Two Enhanced USART • 35 I/O Pins • 10-Bit Analog-to-Digital Converter with Computation (ADCC) 19 CHAPTER 4. ACQUISITION MODULE It is also important to mention that the HPC Development Board has two buttons, four LEDs and a potentiometer to debug and check if the program is working. It also has an Integrated Programmer/Debugger with USB Interface which makes the integration seamless with MPLAB X IDE and Code Configurator and Mikrobus™ support that was used for the Bluetooth Module, which is going to be discussed in section 4.2. The microcontroller programming was based on the photodetector’s datasheet [28]. Figure 18: Signal required by the photodetector In Figure 18, it is possible to see the four signals that are needed to get the read from the photodetector and how they need to be relatively to each other. To accomplish this, four different ports were used to send the signal (as can be seen in Figure 25). To create a similar signal as in Figure 18, three functions were created. A function named tick() was created to control the output of each port. void tick(int times){ int i; for(i=0;i<times;i++){ __delay_us(100); } } tick() uses the assembly function __delay_us() that makes the microcontroller wait the given amount of time in microseconds. A simple function of printing was created using the predefined function EUSART1_Write() , called print() . void print(char* str){ int i; for(i=0;str[i]!='\0';i++){ EUSART1_Write(str[i]); } } 20 4.1. MICROCONTROLLER Another function was created, called read , which can be seen in Appendix A. Its purpose is to do the whole reading process, since the beginning until the testing if the reading is complete. It uses a variable called stop to get the value from End of scan pin. This pin is always at 5𝑉except when the scan is over, which goes to 0𝑉. Since the ADC’s voltage reference is 5𝑉, that means that stop will be always 255 until the scan has ended. Figure 19: Photo of the oscilloscope Before connecting to the photodetector, an oscilloscope was used to test if all four signals were working properly. Figure 19 is a photo of the oscilloscope with the reading signals. Where channel 1is signal 𝜙1, channel 2is signal 𝜙2, channel 3is signal 𝜙𝑠𝑡 and channel 4is signal 𝑅𝑒𝑠𝑒𝑡𝜙. Concluding, Figure 19 is similar to Figure 18. To test if the microcontroller was getting the voltage from the photodetector, a test was made. Figure 20: Graph from 1st test Figure 20 is a graph obtained by making a reading from the photodetector when the smartphone’s 21 CHAPTER 4. ACQUISITION MODULE flashlight is moving closer and then further away three times. And sending the data by serial port to the computer. With this test, it is possible to conclude that the photodetector can detect light and its response is directly proportional and that the microcontroller is receiving the data correctly. 4.2 Bluetooth Module To make the communication between the cellphone and the reader wireless, a bluetooth module was selected with a mikroBUS™ interface and a RN4020 module. Figure 21: BLE2 Click The main feature to select this module was the Microchip Low-energy Data Profile (MLDP) that makes the reader working as a wireless with serial communication protocol. This module can be programmed using the UART serial communication port. To make this process the easiest possible, the USB Interface from the HPC Development Board and a terminal called CoolTerm were used. Figure 22 is a photo of the layout used to program the Bluetooth module. The orange and white cables are for receiving and transmitting pins, respectively. All the red wire are connected to 3,3𝑉and all the blue wire are connected to ground. Figure 22: Photo of setup to program the Bluetooth module 22 4.2. BLUETOOTH MODULE For the purpose of this project, the Bluetooth module’s programming was very simple. First, it was set the baud rate. In this case, the baud rate suggested in the datasheet was used, this is 115200. Second, the needed services were selected. Since only the information from the photodetector is needed on the smartphone or computer, only device information and a private service were selected. Then, a private service is created with two characteristics, one for reading and other for writing. At last, the features of the module are selected, in this case, only Auto Advertisement was selected. In Figure 23, it is possible to see a print screen of CoolTerm and all the commands made to program the Bluetooth module. At the end, an LS command is executed to check if everything was created as expected. Figure 23: Programming of the Bluetooth module 23 CHAPTER 4. ACQUISITION MODULE 4.3 UV LED The UV LED was selected based on two main variables: emission wavelength and angle of emission. Figure 24: MT3650W3-UV Knowing that the Quantum Dots’ excite at 365 𝑛𝑚 and emit at 610 𝑛𝑚, a LED was chosen with the following features. Table 2: Main features of selected UV LED Peak Emission Wavelength Power Output Reverse Current Half Intensity Beam Angle 365 𝑛𝑚 1,5𝑚𝑊 80 𝑚𝐴 ±45◦ 4.4 Circuit Figure 25 shows a schematic of the main components of the project. Looking at the microcontroller, it has 4 pins without connection (RA4 to 7). This is represented in this way because these pins were used only for debugging with the LEDs inlaid in the development board. Then, there are 3 connections to the BLE2 Click using ports RA1, RD0 and RC2. These connections are for waking and change states of the Bluetooth module that was discussed in section 4.2, and are also made through the PCB of the development board. The Bluetooth module has 2 more connections for the serial port communication with the microcontroller. On the right side of the microcontroller, it is possible to see 4 ports dedicated to send the signal to the photodetector (RB0 to 3). And a last pin (RB5) to control the UV LEDs. The photodetector and the microcontroller are connected by two wires. The first is from the End of scan pin to RA3. The purpose of this connection is to tell the microcontroller when the scan is over. The second connects the Active Video pin to RA2, passing through some amplification. An inverting amplifier configuration was chosen to make the photodetector’s output voltage directly proportional to the intensity of light. For this, a rail to rail OpAmp was used, more precisely, an OPA2344. To make the voltage offset, a potentiometer connected to 5𝑉and ground was used. To know what position should the potentiometer be, a simulation in Tina-TI was made. 24 4.5. READER’S CASING 18/10/2022, 15:24 circuit (1).svg file:///Users/elramalho/Downloads/circuit (1).svg 1/1 +5V +5V +5V +5V 10 kΩ 10 kΩ 10 kΩ 10 kΩ 10 kΩ 27 kΩ BLE2 Click CON CMD WKE TX RX 3V3 GND GND S3924-512Q φ2 φ1 φst Vss Vscg Resetφ Reset V Vss Active Video Dummy Video End of scan Vsub Vdd PIC18F47Q10 RA1 RB5 RA2 RA3 RB3 RA4 RB2 RA5 RB1 RB0 TVDD GND TVDD GND RA7 RA6 RC7 RC6 RC2 RD0 Figure 25: Circuit Diagram of major reader’s components Summing up, the Bluetooth module is attached to the development board, where the microcontroller is. Then, the microcontroller sends the signal by four channels to the photodetector. Hereafter, the photodetector sends the signal to the microcontroller, passing through an amplification system with a gain of 2,7 and a voltage offset of 1,25 𝑉. In Figure 26, it is possible to see a photo of the setup working. Here, the UV LED got replaced by a blue LED to test if the system was working. 4.5 Reader’s casing To assemble every component into one and create a first prototype, a team of USN bachelor’s students from Mechanical Engineering was recruited to help creating the case of the reader. Figure 27 shows the final product. In order to create the case, several goals had to be achieved: • The case had to be lighter, easier to handle and more stable; • All the electronic components inside needed to be securely attached; • The reader required a power button and a charging port; • The photodetector needed to be perfectly aligned with the strip’s window. 25 CHAPTER 4. ACQUISITION MODULE Figure 26: Photo of the whole setup Figure 27: Photo of the whole setup The assembling instructions are shown in Figure 28, where Figure 28(b) shows how to assemble the internal structure and place perfectly all the electronic components and Figure 28(a) indicates how to cover the internal structure with an external case. More detailed assembling instructions can be found in Appendix B. 26 4.5. READER’S CASING (a) External Case (b) Internal Case Figure 28: Assembling instructions Another obstacle needed to overcome was the prevention of cross talk between the lines. For this, a filter, inspired in a parallel hole collimator, was created to let only the perpendicular light pass through. Figure 29: Filter’s 3D model Figure 29 shows, from two different points of view, the created filter to prevent cross talking. The four outside holes are for placement to the structure in the reader. The two round holes are for the two UV LEDs placement. And in the middle there is an array of tiny holes, where the photodetector would be and receive the light that it is directly below it. 27 CHAPTER 4. ACQUISITION MODULE Figure 30 represents a schematic of how the filter works and prevents the cross talk. Figure 30: Schematic of cross talk prevention A test was made to verify if the light could pass with a small tilt of the light source. Figure 31 is two pictures from that test. Where the left one is when the light source is perpendicular to the filter and the light can be seen on the table. And the right one is when the light source has a small tilt of approximately 2 degrees and the light cannot be seen below the filter. Figure 31: Filter testing To conclude, the filter works for a normal light source, therefore, it will work with a strip where the light source are quantum dots. 4.6 Python GUI A python program with a graphic user interface (GUI) was created to collect the data and show it to the user in the form of a graph. At a first step, the serial port to receive the data, using the package serial . Figure 32 shows the GUI that uses the serial port to collect the data. At the bottom there are 5 buttons. The first one, Begin , is to start the reading and collecting the data. The Clear button is to clean the graph for a second scan. The third button closes correctly the program. 28 5.2. FUTURE WORK 5.2 Future Work A working prototype was made, giving a value of voltage in response to the light intensity. However, more work could be done to improve the device. The most important, the reader should be tested with strips that have quantum dots lines. Giving the value of minimum and maximum light intensity that the strips can give. Furthermore, a relation between biomarker’s concentration and light intensity, so the final result can be in biomarker’s concentration instead of the value of voltage the ADC reads. Figure 37 shows a possible output from the reader, using a strip with quantum dots as input. 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Thisdocumentwascreatedwiththe(pdf/Xe/Lua)L A T E XprocessorandtheNOVAthesistemplate(v6.10.10)[1].12cc90221730b8ba41bb3b1f8b517acd [1] J.M.Lourenço.TheNOVAthesisL A T E XTemplateUser’sManual.NOVAUniversityLisbon.2021.URL:https://github.com/joaomlourenco/novathesis/raw/master/template.pdf(cit.onp.39). 39 A Programming Code A.1 Microcontroller Code void print(char* str) { int i; for(i=0;str[i]!='\0';i++) { EUSART1_Write(str[i]); } } void tick(int times){ int i; for(i=0;i<times;i++) { __delay_us(100); } } void read() { char str[10]; static uint8_t adcResult; // start 40 A.1. MICROCONTROLLER CODE LATB5 = 0; LATB0 = 0; LATB1 = 0; LATB2 = 1; LATB3 = 1; tick(1); //phi start LATB5 = 1; LATB0 = 1; tick(1); //first impulse LATB2 = 0; LATB1 = 1; tick(1); //end of start LATB0 = 0; tick(1); LATB1 = 0; LATB3 = 0; LATB2 = 1; tick(3); LATB3 = 1; tick(3); int stop = 255; int counter = 0; while(stop>200) 41 APPENDIX A. PROGRAMMING CODE { adcResult = ADCC_GetSingleConversion(PHOTO_CHANNEL) >> 2; stop = ADCC_GetSingleConversion(EOS_CHANNEL) >> 2; print("SHW,0018,"); sprintf(str, "%d", adcResult); print(str); sprintf(str, "%d", counter); print(str); print("\r\n"); counter++; LATB1 = 1; LATB2 = 0; LATA6 = 1; tick(2); LATB3 = 0; LATB1 = 0; LATB2 = 1; LATA6 = 0; tick(2); LATB3 = 1; tick(4); } LATB = 0; print("SHW,0018,000000001111111111111111\r\n"); } void main(void) { SYSTEM_Initialize(); uint8_t n; char str[20]; 42 A.2. COMPUTER CODE RD0 = 1; RC2 = 1; while (1) { if(PIR3bits.RC1IF) { n = EUSART1_Read(); if(n==11) { RC2 = 0; read(); RC1 = 1; } } } } A.2 Computer Code from ast import If import serial import numpy as np import PySimpleGUI as sg import glob import asyncio import bleak def make_vertical(title): new_title = "" for letter in title: new_title += f"{letter}\n" return new_title buttons = [ 43 APPENDIX A. PROGRAMMING CODE [sg.Button(button_text='Begin'), sg.Button(button_text='Close')] ] newdevices = [] deviceslist = [] layout = [ [sg.Text('Scanner', font = ("Arial",30))], [sg.Text('Select device'), sg.Drop(values = newdevices, key='devices', size=20), sg.Button(button_text='Scan for Devices')], ↩→ ↩→ [sg.Text(make_vertical('Intensity'), justification='center', text_color='black'), sg.Graph(canvas_size=(800,450),↩→ graph_bottom_left=(0,0), graph_top_right=(100,100), background_color='white', key='graph')], [sg.Push(), sg.Text('distance (pixels)', justification='center', text_color='black'), sg.Push()],↩→ [sg.Push(), sg.Column(buttons, element_justification='center'), sg.Push()]↩→ ] errorLayout = [ [sg.Text('Please scan for devices first')], [sg.Button(button_text='Dismiss', key='Dismiss')] ] scanLayout = [ [sg.Text('Please choose a device from the list')], [sg.Button(button_text='Dismiss', key='Dismiss')] ] window = sg.Window('Scanner', layout, default_element_size=(20,1), font=("Helvetica",20))↩→ graph = window['graph'] 44 4 x M3 (L= 10mm) ISO7045-M3x10-Z-10S 51