Development of a Low-Cost UV-Vis Spectrophotometer and Its Application for the Detection of Mercuric Ions Assisted by Chemosensors

sensors

Article Development of a Low-Cost UV-Vis Spectrophotometer and Its Application for the Detection of Mercuric Ions Assisted by Chemosensors

David González-Morales 1, Asmilly Valencia 2, Astrid Díaz-Nuñez 3, Marcial Fuentes-Estrada 4, Oswaldo López-Santos 1,* and Olimpo García-Beltrán 4,*

1 Facultad de Ingeniería, Universidad de Ibagué, Carrera 22 Calle 67, Ibagué 730002, Colombia; [email protected] 2 Facultad de Ingeniería Forestal, Universidad del Tolima, Altos de Santa Helena, Ibagué 730001, Colombia; [email protected] 3 Universidad Nacional de Colombia, Sede Medellín, Escuela de Química, Carrera 65, No. 59A-110, Medellín 050034, Colombia; [email protected] 4 Facultad de Ciencias Naturales y Matemáticas, Universidad de Ibagué, Carrera 22 Calle 67, Ibagué 730002, Colombia; [email protected] * Correspondence: [email protected] (O.L.-S.); [email protected] (O.G.-B.); Tel.: +57-8-2760-010 (O.G.-B.) Received: 13 December 2019; Accepted: 5 February 2020; Published: 8 February 2020

Abstract: Detection of an environmental contaminant requires the use of expensive measurement equipment, which limits the realization of in situ tests because of their high cost, their limited portability, or the extended time duration of the tests. This paper presents in detail the development of a portable low-cost spectrophotometer which, by using a specialized chemosensor, allows detection of mercuric ions (Hg2+), providing eﬀective and accurate results. Design speciﬁcations for all the stages assembling the spectrophotometer and the elements selected to build them are presented along with the process to synthesize the chemosensor and the tests developed to validate its performance in comparison with a high-precision commercial laboratory spectrophotometer.

Keywords: water contaminants; low-cost spectrophotometer; Hg2+ ions; chemosensor; coumarin

1. Introduction A spectrophotometer is a measuring device for quantitative analysis generally used to characterize chemical substances by determining the amount of light that is partially absorbed by the analyte present in solution [1]. They can be classified according to the spectral region of work, such as ultraviolet spectrophotometer (UV), from 190 nm to 380 nm; visible spectrophotometer (Vis), from 380 nm to 750 nm; and near infrared spectrophotometer (NIR), from 800 nm to 2500 nm [2–4]. According to their use, they are classified in stationary devices for analysis in laboratories and portable devices for determination of substances in fieldwork [1]. There are spectrophotometers that work in non-visible ranges of the electromagnetic spectrum, such as those presented in References [5,6] (UV and NIR, respectively), which use high-cost elements like diode arrays and charged couple devices (CCD) as sensors in the detecting stage. For the visible range, there are several devices that include a Light Emitting Diode (LED) as radiation source [7], mainly because they have low costs and allow easy implementation. It is worth mentioning the developments presented in References [8,9] which made analyses for three fixed wavelengths and that reported in References [10–12] for seven fixed wavelengths which can be selected by the user with the help of mechanical components. Also, a combination of a white LED and interference filters can be

Sensors 2020, 20, 906; doi:10.3390/s20030906 www.mdpi.com/journal/sensors Sensors 2020, 20, 906 2 of 16 used for obtaining different wavelengths as in Reference [13]. Additionally, some devices are used for chemistry education and allow the user to make a spectral sweep between 400 nm and 700 nm including a rotating diffraction grating [14]. In terms of portable equipment, those implemented in References [15–21] can also make spectral sweeps by means of the integration of small motors, light-scattering elements, low-cost light sources, and simple light detectors. Furthermore, there are commercial and robust devices for use in laboratory such as the Cary 60 UV-Vis (Agilent Technologies, CA, USA) [22], which allow the user to make many types of tests, obtaining high-quality analyses, selectivity, and accuracy at a cost of around 30,000 USD. Portable equipment, such as the DR 1900 (HACH, CO, USA) [23], provides versatility and other advantages in fieldwork at costs between 2000 USD and 8000 USD. Over the last few decades, the impact of heavy metal pollution has been increasing [24]. Mercury is the heavy metal with the greatest impact on the pollution of ecosystems worldwide [25], contributing to bioaccumulation in different organisms and being present in the food chain [26]. Its oxidation state Hg2+ has a high affinity for sulfhydryl groups present in biomolecules such as proteins and endogenous thiols [27,28], which makes it possible to associate the accumulation of this metal to multiple physiological disorders, the best known of which are damages to the kidney, central nervous, immune, and endocrine systems [29–34]. There are a variety of analytical methods for determining mercury; however, the vast majority are complex and require a great deal of resources for implementation, maintenance, and sample analysis. Each of these methods has its own advantages and limitations. Among the best-known methods for determining mercury are hyper Rayleigh scattering (HRS), atomic absorption/emission spectrometry, inductively coupled plasma mass spectrometry (ICP-MS), colorimetric detection, cold vapor atomic fluorescence spectrometry, high-performance liquid chromatography, ion chromatography, and electrochemical sensing [35–44]. During the last decades, there has been increasing interest in developing chemical sensors that can interact with the analyte through desulphurization, chelation, mercury-mediated hydrolysis reactions, and inclusion in macromolecules assisted by chemosensors [24,27,35,45]. However, this tool complemented with low-cost UV-Vis spectrophotometry provides potential alternatives for countries which cannot have access to expensive instrumentation and sophisticated operating processes. This paper presents the development of a portable low-cost spectrophotometer, which by using a chemosensor coumarin-derivate allows detection of mercuric ions (Hg2+) in an aqueous medium. The proposed device has important characteristics such as accuracy, low cost, and portability. The rest of the paper is organized as follows: Section2 presents the components required to build the spectrophotometer, the required embedded digital control algorithms, the methods related with the synthesis of the chemosensors, and the process necessary to perform each test. After that, Section3 presents results obtained using the proposed device in comparison with a commercial device and provides a discussion about how the effectiveness and accuracy of the built device enables its potential real application. Finally, conclusions are presented in Section4.

2. Materials and Methods

2.1. Reagents All analytes were purchased from Sigma-Aldrich and Merck and were used as received. The metal salts used were HgCl CdCl , PbCl , CaCl , MgCl , ZnCl , FeNH (SO ) 12H O, 2, 2 2 2 2 2 4 4 2· 2 Fe-(NH4)2(SO4)2 6H2O, CuCl2, MnCl2, and CoCl2. All solutions employed in this work were · 1 13 prepared in EtOH-H2O (20/80). H and C NMR spectra were recorded on an Avance II 400 MHz multidimensional spectrometer (Bruker Corporation, Billerica, MA, USA), using the solvent or the Tetramethylsilane (TMS) signal as an internal standard. High-resolution mass spectra were recorded on a Pegasus GC GC (Leco Corporation, St. Joseph, MI, USA), GC-MS system with time of ﬂight analyser, × with 40,000 resolution. Absorption spectra were obtained by means of a Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Sensors 2020, 20, 906 3 of 16 Sensors 2020, 20, x FOR PEER REVIEW 3 of 16

2.2.2.2. Chemosensor Development TheThe compound compound was was prepared prepared using using a conventional a conventional three-step three-step synthesis synthesis from commercial from commercial precursors precursorsuntil 7-(diethylamine)-2 until 7-(diethylamine)-2H-chromen-2-oneH-chromen-2-one (2) was obtained, (2) was followed obtained, by followedthionation by with thionation Lawesson´s with Lawessonreagent (Merck-Darmstadt,´s reagent (Merck-Darmstadt, Germany.) Germany.)as reported as reportedin the literature in the literature to obtain to obtain the thecompound compound 7- 7-(diethylamino)-2(diethylamino)-2H-chromene-2-thioneH-chromene-2-thione (3) ([46].3)[46 ]. Synthesis of 7-(diethylamine)-2H-chromen-2-one (2) (9.8692 g, 87.84 %). 1H 1NMR (DMSO-d6) δ 7.50 Synthesis of 7-(diethylamine)-2H-chromen-2-one (2) (9.8692 g, 87.84 %). H NMR (DMSO-d6) δ 7.50(d, J = (d, 8.0J =Hz,8.0 1H), Hz, 7.21 1H), (d, 7.21 J = 8.0 (d, Hz,J = 1H),8.0 Hz, 6.53 1H), (dd, 6.53 J = 12.0 (dd, Hz,J = J 12.0= 2.0 Hz,Hz, J1H),= 2.0 6.44 Hz, (d, 1H), J = 2.0 6.44 Hz, (d, 1H);J = 5.992.0 (d, J = 8.0 Hz, 1H), 3.42 (q, J = 8.0 Hz, 4H), 1.17 (t, J = 8.0 Hz, 6H). 13C NMR (DMSO-13d6): 162.7, 157.1, 151.1, Hz, 1H); 5.99 (d, J = 8.0 Hz, 1H), 3.42 (q, J = 8.0 Hz, 4H), 1.17 (t, J = 8.0 Hz, 6H). C NMR (DMSO-d6): 162.7,144.1, 157.1,129.2, 109.5, 151.1, 109.07, 144.1, 129.2,108.5, 109.5,97.9, 45.2, 109.07, 12.8. 108.5, 97.9, 45.2, 12.8. Synthesis of 7-(diethylamino)-2H-chromene-2-thione (3) (1.455 g, 62.47%). 11H NMR (DMSO-d6) δ Synthesis of 7-(diethylamino)-2H-chromene-2-thione (3) (1.455 g, 62.47%). H NMR (DMSO-d6) δ 7.31–7.29 (m, 2H), 6.96 (d, J = 12, 1H), 6.66–6.64 (m, 2H).13 13C NMR (DMSO-d6): 197.8, 159.6, 151.3 136.1, 7.31–7.29 (m, 2H), 6.96 (d, J = 12, 1H), 6.66–6.64 (m, 2H). C NMR (DMSO-d6): 197.8, 159.6, 151.3 136.1, 128.9,128.9, 123.3,123.3, 110.8,110.8, 110.0,110.0, 97.3,97.3, 45.0,45.0, 12.4. 12.4. High-resolutionHigh-resolution electrosprayelectrospray ionisationionisation massmass spectrometryspectrometry (HRESIMS) was consistent with the molecular formula of C9H14OS (required: m/z obsd 233.0868; calcd for (HRESIMS) was consistent with the molecular formula of C9H14OS (required: m/z obsd 233.0868; calcd for233.0869). 233.0869).

2.3.2.3. Determining the Quantum Yield ofof EmissionEmission FluorescenceFluorescence quantumquantum yieldyield ofof 33 waswas measuredmeasured usingusing aa solutionsolution ofof RhodamineRhodamine 101101 inin ethanolethanol asas standardstandard ((ΦΦss == 1). All All values values were corrected considering the solvent refractionrefraction index.index. QuantumQuantum yieldyield waswas calculatedcalculated usingusing EquationEquation (1), where the subscripts x and s denote sample and standard, respectively;respectively; φФ isis thethe quantumquantum yield;yield; ηη isis thethe refractiverefractive index;index; andand GradGrad isis thethe slopeslope fromfrom thethe plotplot ofof integratedintegrated fluorescencefluorescence intensityintensity vs.vs. absorbanceabsorbance [[35].35]. Φ =Φ! 2! Grad x ηx (1) Φ = Φ (1) x s 2 Grads ηs 2.4. Detection Limit 2.4. Detection Limit The detection of limit (LOD) was calculated based on absorbance and fluorescence titrations. To The detection of limit (LOD) was calculated based on absorbance and fluorescence titrations. determine the signal to noise ratio, the absorbance (at 388 nm) of 3 with Hg2+ was measured three To determine the signal to noise ratio, the absorbance (at 388 nm) of 3 with Hg2+ was measured times and the standard deviation of calibration curve was determined. The detection limit was three times and the standard deviation of calibration curve was determined. The detection limit was calculated with the equation LOD = 3 σb/m, where σb is the standard deviation of calibration curve calculated with the equation LOD = 3 σ /m, where σ is the standard deviation of calibration curve and m is the slope of the plot of absorbanceb or fluorescenceb intensity versus analyte concentration and m is the slope of the plot of absorbance or fluorescence intensity versus analyte concentration [35]. [35]. 2.5. Electronic Components 2.5. Electronic Components The proposed low-cost spectrophotometer works in the visible part of the electromagnetic spectrum, fromThe 400 nmproposed to 700 nm low-cost (see Figure spectrophotometer1). It is composed works by a low-power in the visible light source,part of a the monochromator electromagnetic for selectingspectrum, specific from 400 wavelengths, nm to 700 and nm two (see light-isolated Figure 1). It sample is composed chambers. by a Each low-power sampling light chamber source, has a anmonochromator independent detector for selecting which specific allows thewavelengths, spectrophotometer and two tolight-isolated make measurements sample chambers. of the reference Each andsampling the studied chamber substance has an in independent a single experiment. detector which allows the spectrophotometer to make measurements of the reference and the studied substance in a single experiment.

Figure 1. System architecture of the developed spectrophotometer.

2.5.1. Light Source

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, 906 4 of 16

2.5.1. Light Source Light Emission Diodes (LEDs) are widely used as light sources in analytical instrumentation applications [47]. For the proposed device, it is important to use a light source that emits radiation in the visible region of the electromagnetic spectrum. A 1-W low-cost LED with cold white color with a 5◦ collimator lens was selected, allowing the radiation pattern to be concentrated and reﬂected in a small portion of the diﬀraction grating, reducing any light power while the test is made. The relative intensity of a white LED shows that it has a good response in the visible region. Also, by using the additional hardware presented in Figure1, the LEDs provide a practical way to have an economic alternative for the radiation source with low energy consumption (Supplementary Materials Figure S1).

2.5.2. Monochromator The main task of the monochromator is to select and transmit one specific wavelength of the visible spectrum to the sample. This goal is achieved in the proposed device using a National Electrical Manufacturers Association (NEMA) 17 hybrid stepper motor in micro-stepping configuration together with a low-cost diffraction grating and a small slit that is used for selecting wavelengths. The angle displacement of the diffraction grating is performed in steps of 1.8◦, resulting in 200 steps for a full 360-degree turn. The Allegro a4988 controller is used in micro-stepping configuration to obtain a good resolution for sweeping all the wavelengths of the visible spectrum enlarging the number of steps. As the driver makes 1/16 of a step, the resolution results in 0.1125◦ per step. For details of the configuration of the driver, see Supplementary Materials Figure S2. The diffraction grating splits and diffracts the radiation emitted by the white LED into individual wavelengths [48]. Unlike other dispersive elements such as mirrors and prisms, diffraction gratings are elements with good performance and lower costs. These are composed of a large number of small parallel slits that have equal spacing between them along the grating and which are capable of diffracting the incident light that falls on them. Depending on the overtone and the incident angle of the light beam of the source projected over the grating, an individual color can be observed. In the developed system, a diffraction grating obtained from a Digital Versatile Disc (DVD) without information is used. This is a low-cost solution, which contains grooves with a total of 1350 lines/mm and a separation between them of 780 nm, properties that provide good dispersion and resolution (see Table1).

Table 1. Grating separation for diﬀerent diﬀraction gratings.

Grating Separation Grating Lines (inch) Lines (mm) (nm) CD 15,875 625 1600 DVD 34,300 1350 740 Lab grade diﬀraction grating 34,300 1000 1000

The diffraction grating is located in such a way that the range of displacement angles reflects in the samples the components corresponding to the third overtone produced by the reflection effect (Figure2) with the aim of providing a greater spectral resolution in the visible range. Using the micro-stepping configuration available in the a4988 driver, in which one step is reduced to an advance of 0.1125◦, the 300-nm spectrum is converted into 198 steps, resulting in a selectivity for the monochromator of 1.52 nm per step. To find the wavelength of a specific color, the following expression is used (Fraunhofer equation): nλ = d sin θ, (2) where λ is the wavelength of a specific color; n = 1 (grating order); d = 740 (distance between slits in a DVD); and θ = diffraction angle. Sensors 2020, 20, x FOR PEER REVIEW 5 of 16 Sensors 2020, 20, 906 5 of 16

Figure 2. SelectedSelected overtone produced by the diffraction diﬀraction grating.

Table2 shows the values of the incident angle in the di ﬀraction grating for diﬀerent colors.

TableTable 2. IncidentIncident angle angle in the diffraction diﬀraction gratinggrating for obtaining didifferentﬀerent colors.colors.

ColorColor Wavelength Wavelength (nm) (nm) Deflection angle Deﬂection (°) Angle (◦) redred 650 650 61 61 orangeorange 600 600 54 54 yellow 575 51 yellow 575 51 green 550 48 blue-greengreen 550 500 48 42.5 blueblue-green 500 450 42.5 37.5 violetblue 450 400 37.5 32.7 violet 400 32.7 2.5.3. Detector 2.5.3. Detector The main function of a detector is to convert a light signal into an electric signal [49]. The TEMT6000 ambientThe lightmain sensorfunction (Vishay of a detector Intertechnologies, is to convert PA, a USA) light issignal used into for thean detectionelectric signal stage. [49]. This The is aTEMT6000 surface mount ambient device light that sensor is composed (Vishay Intertechnologi of an Negativees, Positive PA, USA) Negative is used (NPN)for the phototransistordetection stage. mountedThis is a on surface a printed mount circuit device board (PCB)that is of 1composed cm 1 cm. of Its an relative Negative spectral Positive response Negative is similar (NPN) to the × humanphototransistor eye, having mounted good on sensitivity a printed in circuit the visible board range(PCB) ofof the1 cm electromagnetic × 1 cm. Its relative spectrum spectral (Figure response3). Theis similar analog to voltage the human output eye, is proportional having good to thesensitivity received in amount the visible of light. range This of voltage the electromagnetic is conditioned beforespectrum being (Figure sent to3). theThe microcontroller, analog voltage usingoutput a non-invertingis proportional ampliﬁer to the received of gain amount 5. The detectorsof light. This are locatedvoltage is in conditioned such a way thatbefore they being can sent receive to the the microcontroller, resulting monochromatic using a non-inverting radiation after amplifier having of passedgain 5. throughThe detectors the sampleare located and in reference such a way substances that they storedcan receive in the the quartz resulting cuvettes. monochromatic Each position radiation covered after byhaving the monochromatorpassed through the corresponds sample and toreference a level substances of voltage stored in the in detector, the quartz proportional cuvettes. Each to the position light transmittedcovered by the through monochromator the reference corresponds and sample to a studied level of substances.voltage in the detector, proportional to the light transmitted through the reference and sample studied substances.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, 906x FOR PEER REVIEW 66 of of 16

Figure 3. Details of the detector stage. Figure 3. Details of the detector stage. Based on Beer Lambert’s law, the concentration value of a chemical substance is directly proportionalBased on to Beer the amount Lambert’s of light law, it the absorbs concentratio when exposedn value to of monochromatic a chemical substance radiation. is Oncedirectly the proportionalvoltage measurements to the amount corresponding of light it absorbs to the light when captured exposed in to the monochromatic detector for the radiation. reference Once and the voltagesubstance measurements being studied corresponding are obtained, theto the following light ca expressionptured in the is used detector to ﬁnd for the the absorbance reference and value: the substance being studied are obtained, the following expression is used to find the absorbance value: P A = log10P = log10(T) (3) A=log Po = −log− (T) (3) Po where A is absorbance; P is magnitude of transmitted light through the sample; Po is magnitude of where A is absorbance; P is magnitude of transmitted light through the sample; Po is magnitude of transmitted light through the reference; and P/Po = T, which is transmittance. transmitted light through the reference; and P/Po = T, which is transmittance. Using voltage levels, we have Using voltage levels, we have ! P Vsample A = log P = logVsample (4) A=log 10 = −log (10 ) (4) P0P0 − V solventV solvent Table3 3 presents presents thethe most most absorbed absorbed color color in in every every produced produced wavelength. wavelength.

Table 3. Light absorbance for wavelengths in visible spectra.

WavelengthWavelength Absorbed Absorbed color Color Solution color Solution Color 380–435380–435 Violet Violet Green-yellow Green-yellow 435–480435–480 Blue Blue Yellow Yellow 480–490480–490 Blue-green Blue-green Orange Orange 490–500490–500 Green-blue Green-blue Red Red 500–560 Green Violet 500–560 Green Violet 560–580 Green-yellow Violet 580–595560–580 Green-yellow Yellow Violet Blue 595–650580–595 Yellow Orange Blue Blue-green 650–780595–650 Orange Red Blue-green Green-blue 650–780 Red Green-blue 2.5.4. Microcontroller and Control Algorithms 2.5.4. Microcontroller and Control Algorithms An Atmega 138p microcontroller (Atmel Corp., San Jose, CA, USA) with an Arduino interface wasAn used Atmega to implement 138p microcontroller the control (Atmel algorithms Corp., required CA, USA) by with the an spectrophotometer Arduino interface operation.was used toFirst, implement an initialization the controlroutine algorithms is performed, required which by consists the spectrophotometer in finding an initial operation. position First, for thean initializationmonochromator routine and using is aperformed, physical reference which withconsists a limit in switch finding that guaranteesan initial thatposition all tests for start the at monochromatorthe same wavelength. and usingThen, a usingphysical micro-stepping reference with, the a limit stepper switch motor that sweeps guarantees all the that wavelengths all tests start of atthe the visible same spectrum. wavelength. In each Then, step, using the micro-steppi voltage valueng, produced the stepper by themotor detector sweeps is obtained,all the wavelengths measuring ofthe the resulting visible monochromaticspectrum. In each light step, that the has voltage passed throughvalue produced the sample. by Thisthe detector voltage isis read obtained, by the measuringanalog-to-digital the resulting converter monochromatic of the microcontroller, light wherethat has it ispassed filtered through using a simplethe sample. average This digital voltage filter. Afteris read that, by the the analog-to-digital absorbance value converter is computed of the and mi storedcrocontroller, in a table. where The flowchartit is filtered of using the developed a simple averagealgorithm digital is presented filter. inAfter Figure that,4. the absorbance value is computed and stored in a table. The flowchart of the developed algorithm is presented in Figure 4.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, 906 7 of 16 Sensors 2020, 20, x FOR PEER REVIEW 7 of 16

Figure 4. Flowchart of the microcontroller algorithm. Figure 4. Flowchart of the microcontroller algorithm. 2.5.5. Detailed Hardware Setup 2.5.5. Detailed Hardware Setup The components are assembled in a black box made with polylactic acid (PLA) polymer by means of 3DThe printing components with a sizeare ofassembled 15 cm in22 a cm black8 box cm andmade a wallwith andpolylactic top cover acid thickness (PLA) polymer of 4 mm, by × × idealmeans to reduceof 3D printing the incidence with a ofsize external of 15 cm light × 22 interferences. cm × 8 cm and In a order wall and to obtain top cover good thickness light dispersion, of 4 mm, theideal diﬀ toraction reduce grating the incidence is located of external 18 cm from light the interf sampleerences. chambers. In order In to addition, obtain good it has light the dispersion, ability to rotatethe diffraction its position grating to make is located incident 18 angles cm from between the sample 25◦ and chambers. 70◦, which In guarantees addition, it the has generation the ability of to monochromaticrotate its position light to betweenmake incident 400 nm angles and 700 between nm. The 25°detectors and 70°, which are in aguarantees room with the light generation insulation of thatmonochromatic reduces the interference light between in the 400 experiments nm and 700 and nm. guarantees The detectors zero are voltage in a whenroom with the radiation light insulation source isthat oﬀ. reduces Figure5 showsthe interference the physical in distributionthe experiments of the and components. guarantees zero voltage when the radiation source is off. Figure 5 shows the physical distribution of the components.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, 906 8 of 16 Sensors 2020, 20, x FOR PEER REVIEW 8 of 16

(a)

(b)

FigureFigure 5. 5.( a()a Geometric) Geometric distribution distribution of of the the spectrophotometer spectrophotometer hardware; hardware; (b ()b dimensions) dimensions of of the the developeddeveloped system. system. 2.5.6. Tests Applied to the Developed System 2.5.6. Tests Applied to the Developed System To test the operation of the developed equipment, an initial test is performed to measure the To test the operation of the developed equipment, an initial test is performed to measure the concentration of the sensor with coumarin, as is done in Reference [50]. A solution with ethanol is used concentration of the sensor with coumarin, as is done in Reference [50]. A solution with ethanol is as reference, and 4 solutions with coumarin in 4 known concentrations are tested: 5 µM, 10 µM, 15 µM, used as reference, and 4 solutions with coumarin in 4 known concentrations are tested: 5 μM, 10 μM, and 20 µM. The second test consists in measuring mercury concentration. Diﬀerent solutions are 15 μM, and 20 μM. The second test consists in measuring mercury concentration. Different solutions prepared with known concentrations of 1 µM, 2 µM, 3 µM, and 4 µM, which are added to the coumarin are prepared with known concentrations of 1 μM, 2 μM, 3 μM, and 4 μM, which are added to the with the sensor. Figure6 shows the color change e ﬀect in coumarin when detecting mercury. The tests coumarin with the sensor. Figure 6 shows the color change effect in coumarin when detecting run in parallel, making comparisons between the results obtained with the developed prototype and mercury. The tests run in parallel, making comparisons between the results obtained with the a commercial Cary 60 spectrometer (Agilent Technologies, Santa Clara, CA, USA). developed prototype and a commercial Cary 60 spectrometer (Agilent Technologies, CA, USA).

3. Results and Discussion When building the low-cost UV-Vis spectrophotometer, consideration was given to the development of a chemosensor to evaluate the optical capabilities of the proposed equipment, which led to a search in the literature to find alternatives for its synthesis. According to that, the developed

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, x FOR PEER REVIEW 9 of 16 chemosensor must have the characteristic to react and determine the metal in aqueous medium by colorimetric and spectrophotometric methods. Compound 3 was prepared in three synthetic steps (Supplementary Materials Scheme S1). 4- (diethylamino)-2-hydroxybenzaldehyde (1) was condensed with diethyl malonate in a Knoevenagel condensation and cyclized and decarboxylated in one step to afford 7-(diethylamino)-2H-chromen-2-one (2). Then, the compound was modified with Lawesson’s reagent (thionation agent) to obtain the

Sensorscompound 2020, 20 7-(diethylamino, x FOR PEER REVIEW)-2-chromene-2-thione (3) (Figure 6) [46]. The final product was characterized9 of 16 Sensorsby using2020 ,120H, 906and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and High Resolution 9Mass of 16 chemosensorSpectrometry (HRMS)must have (Supplementary the characteristic Materials to reac Figurest and S3–S7).determine the metal in aqueous medium by colorimetric and spectrophotometric methods. Compound 3 was prepared in three synthetic steps (Supplementary Materials Scheme S1). 4- (diethylamino)-2-hydroxybenzaldehyde (1) was condensed with diethyl malonate in a Knoevenagel condensation and cyclized and decarboxylated in one step to afford 7-(diethylamino)-2H-chromen-2-one (2). Then, the compound was modified with Lawesson’s reagent (thionation agent) to obtain the compound 7-(diethylamino)-2-chromene-2-thione (3) (Figure 6) [46]. The final product was characterized by using 1H and Figure13C Nuclear 6. Chemical Magnetic s structuretructure Resonance of 7-(diethylamino)-2 (NMR) spectroscopyH-chromene-2-thione. and High Resolution Mass Spectrometry (HRMS) (Supplementary Materials Figures S3–S7). 3. Results and Discussion The main photophysical constants of compound 3 were determined, showing an excitation band (λex) Whenat 483 nm building (λex) and the an low-cost emission UV-Vis band ( spectrophotometer,λex) at 510 nm (Supplementary consideration Materials was givenFigure to S8); the a developmentmolar extinction of a coefficient, chemosensor which to evaluate is 15,754 the M optical-1 cm-1 capabilities(Supplementary of the Materials proposed Figure equipment, S9); awhich quantum led toyield a search of 0.013; in theand literaturea Stokes shift to find of 27 alternatives nm. for its synthesis. According to that, the developed chemosensorCompound must 3 was have tested the characteristic with different to metals react and (Hg determine2+, Cd2+, Pb the2+, Ca metal2+, Mg in2+ aqueous, Zn2+, Fe2+ medium, Fe3+, Cu by2+, colorimetricMn2+, and Co and2+). When spectrophotometric testing metals, methods. it is observed that only mercuric ions react with chemosensor 3 veryCompound quickly andFigure3 was selectively, 6. prepared Chemical showing instructure three a of syntheticcolorime 7-(diethylamino)-2tric steps (Figure (SupplementaryH-chromene-2-thione. 7a), fluorimetric Materials (Figure Scheme 7b), and S1). 4-(diethylamino)-2-hydroxybenzaldehydespectrophotometric UV-Vis (Figure 8). In reviewing (1) was condensed the phenomenon with diethyl that malonateoccurred in in the a Knoevenagel latter result condensationbeingThe notorious, main and photophysical by cyclized adding and mercuric constants decarboxylated ions of in compound the in solution one step 3 wereof to compound aff determined,ord 7-(diethylamino)-2 3, there showing is a decrease anH -chromen-2-oneexcitation in the bandband (of2λ). ex483) at Then, nm 483 and nm the a ( compoundλbathochromicex) and an wasemission displacement modified band with(toλ ex513) at Lawesson’s nm; 510 then, nm (Supplementaryover reagent time, (thionationit is observed Materials agent) that Figure the to 513-nm obtainS8); a molartheband compound decreases extinction and 7-(diethylamino)-2-chromene-2-thionecoefficient, that there which is a hypochromatic is 15,754 M-1 cm shift-1 (Supplementary with (3) an (Figure increase6 )[Materials of46 one]. Theband Figure final in 388S9); product nma quantum UV-Vis was yieldcharacterized[46] (Supplementaryof 0.013; and by using a Stokes Materials1H shift and of 13Figure C27 Nuclearnm. S10) Magnetic and an Resonanceisosbestic (NMR)point at spectroscopy 432. Using and different High -9 ResolutionconcentrationsCompound Mass of 3 Spectrometrymercuric was tested ions with (HRMS)(0.25 different to 2.5 (Supplementary μM), metals the detection(Hg2+ Materials, Cd limit2+, Pb was Figures2+, Ca calculated2+, S3–S7).Mg2+, Zn as2+ 1.1, Fe ×2+ 10, FeM3+, andCu2+ a, Mnlimit2+,The ofand quantification main Co2+). photophysical When wastesting calculated constants metals, itas of is 3.7compound observed × 10-9 M. that3 Inwere onlyaddition, determined, mercuric we emphasize ions showing react with anthat, excitation chemosensorwhen testing band (3withλ exvery) atthe 483quickly addition nm ( λandex of) and mercuricselectively, an emission and showing other band ions ( λaex colorime)simultaneously, at 510 nmtric (Supplementary (Figure no interference 7a), fluorimetric Materials was Figureobserved. (Figure S8); 7b),a molar and 1 1 spectrophotometricextinctionTo confirm coefficient, and UV-Vis elucidate which (Figure is 15,754what 8) was M. In− reviewinghappeningcm− (Supplementary the in thephenomenon reaction, Materials we that conducted Figureoccurred S9); anin a theexperiment quantum latter result yield for beingofthe 0.013; 13C notorious, NMR and (Supplementary a Stokes by adding shift mercuric of 27Materials nm. ions Figure in the S11).solution When of compound compound 3 3, therewas performed is a decrease in thein the absence band 2+ 2+ 2+ 2+ 2+ 2+2+ 2+ 3+ ofand 483 presenceCompound nm and of a mercuricbathochromic3 was tested ions, withadisplacement desulphurization different to metals 513 reaction nm; (Hg then, was, Cd over evident, Pbtime, [35]., Cait is By observed, Mgadding, Zn Hgthat ,,the Fethe 513-nm OC, Fe = S, bandCucarbon2+ ,decreases Mn atom2+, andC2 and of Co thatthe2+ ).therethiocoumarin When is a testinghypochromatic (δ metals,= 194.7) shift itwas is with observedclearly an increasedisplaced that onlyof oneto mercuric 166.0 band ppm,in ions388 a nm reacttypical UV-Vis with C2 [46]chemosensorcarbonyl (Supplementary (OC 3 = very O) signal quickly Materials of anda coumarin selectively, Figure (Supplementary S10) showing and a colorimetrican Materialsisosbestic (Figure Figure point7 a), S10).at fluorimetric 432. Using (Figure different7b), concentrationsand spectrophotometric of mercuric UV-Vis ions (0.25 (Figure to 2.58). μ InM), reviewing the detection the phenomenonlimit was calculated that occurred as 1.1 × in10-9 the M latterand a limitresult of being quantification notorious, was by adding calculated mercuric as 3.7 ions × 10 in-9 M. the In solution addition, of compoundwe emphasize3, there that, is when a decrease testing in withthe band the addition of 483 nm of and mercuric a bathochromic and other displacement ions simultaneously, to 513 nm; no then, interference over time, was it isobserved. observed that the 513-nmTo bandconfirm decreases and elucidate and that what there was is a happening hypochromatic in the shift reaction, with an we increase conducted of one an bandexperiment in 388 nmfor theUV-Vis 13C NMR [46] (Supplementary(Supplementary Materials Figure Figure S11). S10) When and an compound isosbestic 3 pointwas performed at 432. Using in the di absencefferent andconcentrations presence of of mercuric mercuric ions, ions a (0.25desulphurization to 2.5 µM), the reaction detection was limitevident was [35]. calculated By adding as 1.1 Hg2+,10 the9 OCM and = S × − carbona limit ofatom quantification C2 of the wasthiocoumarin calculated ( asδ = 3.7 194.7)10 was9 M. clearly In addition, displaced we emphasize to 166.0 ppm, that, whena typical testing C2 × − withcarbonyl the addition(OC = O) of signal mercuric of a coumarin and other (Supplementary ions simultaneously, Materials no interference Figure S10). was observed.

(a) (b)

Figure 7. (a) Colorimetric change of compound 3 in the presence of mercuric ions; (b) fluorimetric change of compound 3 in the presence of mercuric ions.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors

(a) (b)

Figure 7. ((a)) Colorimetric change of compound 3 in the presence of mercuric ions; (b) ﬂuorimetricfluorimetric change of compound 3 in the presence ofof mercuricmercuric ions.ions.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, 906 10 of 16 Sensors 2020, 20, x FOR PEER REVIEW 10 of 16

Sensors 2020, 20, x FOR PEER REVIEW 10 of 16

Figure 8. Changes in the absorbance spectra of 3 (5 10 6 M) upon addition of ions (1 10 4 M) in Figure 8. Changes in the absorbance spectra of 3 (5× × 10− -6 M) upon addition of ions (1× × 10− -4 M) in H O:EtOH 80:20. H22O:EtOH 80:20. To conﬁrm and elucidate what was happening in the reaction, we conducted an experiment for By validating the response of the Cary 60 spectrophotometer (Agilent Technologies, CA, USA) the 13C NMR (Supplementary Materials Figure S11). When compound 3 was performed in the absence and theFigure built 8. Changeslow-cost in spectrophotometer, the absorbance spectra using of 3 (5both × 10 -6equipment, M) upon addition the length of ions of (1 absorption × 10-4 M) in was and presence of mercuric ions, a desulphurization reaction was evident [35]. By adding Hg2+, the OC determinedH2O:EtOH reporting 80:20. a maximum of at 483 nm. In Figure 9, it can be clearly seen that the absorption = S carbon atom C2 of the thiocoumarin (δ = 194.7) was clearly displaced to 166.0 ppm, a typical C2 bands are very similar between both instruments. This gives a clear idea of the approach in the correct carbonyl (OC = O) signal of a coumarin (Supplementary Materials Figure S10). operationBy validating of the built the device. response Although of the Cary the respon60 spectrophotometerse presented by (Agilent the low-cost Technologies, spectrophotometer CA, USA) By validating the response of the Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, hasand some the builtnoise low-costcompared spectrophotometer, to the Cary 60 spectrop usinghotometer both equipment, (Agilent Technothe lengthlogies, of CA, absorption USA) and was it CA, USA) and the built low-cost spectrophotometer, using both equipment, the length of absorption shoulddetermined be further reporting reduced, a maximum this ofwill at 483imply nm. thatIn Figure additional 9, it can electronic be clearly circuitryseen that theor absorptionadditional was determined reporting a maximum of at 483 nm. In Figure9, it can be clearly seen that the absorption computationalbands are very requirementssimilar between have both a direct instruments. impact onThis th givese cost a of clear the ideadevice of orthe the approach duration in ofthe the correct test, bands are very similar between both instruments. This gives a clear idea of the approach in the correct respectively.operation of the built device. Although the response presented by the low-cost spectrophotometer operationhas some ofnoise the builtcompared device. to the Although Cary 60 the spectrop responsehotometer presented (Agilent by the Techno low-costlogies, spectrophotometer CA, USA) and it hasshould some be noise further compared reduced, to the this Cary will 60 spectrophotometerimply that additional (Agilent electronic Technologies, circuitry Santa or Clara,additional CA, USA)computational and it should requirements be further0,60 reduced,have a direct this willimpact imply on thatthe cost additional of the device electronic or the circuitry duration or additional of the test, computational requirements have a direct impact on the cost of the device or the duration of the respectively. 2 3 [20 μM] test, respectively. 1 0,45 0,60 2 3 [15 μM] 2 1 3 [20 μM] 0,30 1 0,45 2 3 [10 μM] 1

Absorbance μ 0,15 2 3 [15 M] 1 0,30 2 3 [5 μM] 1 2 3 [10 μM] 0,00 1 Absorbance 0,15 400 450 500 550 Wavelength2 (nm) 3 [5 μM] 1 0,00 Figure 9. Comparison of the absorbance400 responses 450 of compound 500 3 using 550 (1) the spectrophotometer Cary 60 and (2) the low-cost spectrophotometer in H2O:EtOH 80:20. Wavelength (nm)

FigureIn contrast, 9. Comparison Table 4 shows of the absorbancethe comparison responses between of compound the measurements3 using (1) the made spectrophotometer using both devices.

It is CaryimportantFigure 60 and9. Comparison to (2 )note the low-cost the ofabsorbance the spectrophotometer absorbance measuremen responses in H2ts O:EtOHof with compound error 80:20. percentages3 using (1) the lower spectrophotometer than 10%, which are similarCary 60 in and both (2) theinstruments. low-cost spectrophotometer The measurement in H 2withO:EtOH the 80:20. error percentage higher than 10% is obtained in the lowest coumarin concentration, which is seen as an opportunity to improve the sensitivityIn contrast, of the Tabledetectors 4 shows so that the they comparison have better between performances the measurements at these specific made conditions.using both devices. It is important to note the absorbance measurements with error percentages lower than 10%, which Sensorsare similar 2020, 20 in, x; doi:both FOR instruments. PEER REVIEW The measurement with the error percentagewww.mdpi.com/jou higher thanrnal/sensors 10% is obtained in the lowest coumarin concentration, which is seen as an opportunity to improve the sensitivity of the detectors so that they have better performances at these specific conditions.

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, 906 11 of 16

In contrast, Table4 shows the comparison between the measurements made using both devices. It is important to note the absorbance measurements with error percentages lower than 10%, which are similar in both instruments. The measurement with the error percentage higher than 10% is obtained in the lowest coumarin concentration, which is seen as an opportunity to improve the sensitivity of the Sensors 2020, 20, x FOR PEER REVIEW 11 of 16 detectors so that they have better performances at these speciﬁc conditions. Table 4. Measurements of coumarin concentration. Table 4. Measurements of coumarin concentration. Absorbance Absorbance Cary Measurement Error percentage Concentration Absorbance Absorbance Cary Measurement Error Percentage Concentration prototypePrototype 60 60 differenceDiﬀerence (%)(%) 5 μM 0.109 0.095 0.014 12.84 5 µM 0.109 0.095 0.014 12.84 1010 μµMM 0.232 0.232 0.226 0.226 0.006 0.006 2.65 2.65 1515 μµMM 0.385 0.385 0.360 0.360 0.025 0.025 6.94 6.94 2020 μµMM 0.532 0.532 0.532 0.532 0.000 0.000 0.0 0.0

With the obtained results, the calibration curve of the instrument for the case of coumarin concentrationWith the obtainedis computed. results, This theis presented calibration in Figure curve of10. the instrument for the case of coumarin concentration is computed. This is presented in Figure 10.

Figure 10. Calibration curve for coumarin concentration. Figure 10. Calibration curve for coumarin concentration. Just like the previous test, both the prototype and the commercial device show the same absorption bandJust characteristics like the previous as compound test, 3both; by adding the protot differentype concentrationsand the commercial of mercuric device ions, show the increasingthe same behaviorabsorption of band a new characteristics absorption bandas compound is evident. 3; by This adding displacement different concentrations occurs by a desulphurization of mercuric ions, reactionthe increasing [35] when behavior Hg2+ reactsof a new with absorption the sulfur ofband thiocarbonyl is evident. of This compound displacement3, additionally occurs by and a perceptibledesulphurization changing reaction to the human[35] when eye Hg given2+ reacts colorimetric with the changes sulfur of of yellow thiocarbonyl to pink. of Likewise, compound it can 3, beadditionally seen in Figure and perceptible11 how the signalchanging forms to the are human very similar eye given between colorimetric the reported changes in bothof yellow instruments. to pink. ThereLikewise, are someit can variationsbe seen in thatFigure are 11 identified how the in signal the lower forms part are ofvery the similar signal between given by the the reported prototype, in whichboth instruments. can be corrected There by are implementing some variations additional that ar filters.e identified in the lower part of the signal given by the prototype, which can be corrected by implementing additional filters.

(a) (b)

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, x FOR PEER REVIEW 11 of 16

Table 4. Measurements of coumarin concentration.

Absorbance Absorbance Cary Measurement Error percentage Concentration prototype 60 difference (%) 5 μM 0.109 0.095 0.014 12.84 10 μM 0.232 0.226 0.006 2.65 15 μM 0.385 0.360 0.025 6.94 20 μM 0.532 0.532 0.000 0.0

With the obtained results, the calibration curve of the instrument for the case of coumarin concentration is computed. This is presented in Figure 10.

Figure 10. Calibration curve for coumarin concentration.

Just like the previous test, both the prototype and the commercial device show the same absorption band characteristics as compound 3; by adding different concentrations of mercuric ions, the increasing behavior of a new absorption band is evident. This displacement occurs by a desulphurization reaction [35] when Hg2+ reacts with the sulfur of thiocarbonyl of compound 3, additionally and perceptible changing to the human eye given colorimetric changes of yellow to pink. Likewise, it can be seen in Figure 11 how the signal forms are very similar between the reported in Sensorsboth instruments.2020, 20, 906 There are some variations that are identified in the lower part of the signal12 given of 16 by the prototype, which can be corrected by implementing additional filters.

Sensors 2020, 20, x FOR PEER REVIEW 12 of 16 (a) (b)

FigureFigure 11. 11.Absorbance Absorbance the the compound compound3 3and and response response in in front front mercuric mercuric ions: ions: ( a()a spectrophotometer) spectrophotometer ®® CaryCary 60; 60; ((bb)) thethe low-costlow-cost spectrophotometerspectrophotometer in in H H22O:EtOHO:EtOH 80:20.80:20. Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors TableTable5 shows5 shows the the comparison comparison between between the the results results obtained obtained from from both both compared compared devices. devices. PercentagePercentage errors errors in in the the absorbance absorbance measurements measurements are are less less than than 5%, 5%, which which shows shows the the accuracy accuracy in in the the detectiondetection of of the the concentration concentration of of mercury mercury ions. ions.

TableTable 5. 5.Measurements Measurements of of mercury mercury concentration. concentration.

AbsorbanceAbsorbance AbsorbanceAbsorbance Cary Cary MeasurementMeasurement ErrorError Percentagepercentage ConcentrationConcentration prototypePrototype 60 60 differenceDiﬀerence (%)(%) 11 μµM 0.066 0.066 0.065 0.065 0.001 0.001 1.54 1.54 22 μµM 0.128 0.128 0.132 0.132 0.004 0.004 3.03 3.03 µ 33 μM 0.162 0.162 0.169 0.169 0.007 0.007 4.14 4.14 4 µM 0.195 0.194 0.001 0.52 4 μM 0.195 0.194 0.001 0.52

WithWith the the obtained obtained results,results, thethe calibrationcalibration curvecurve ofof thethe instrumentinstrument forfor thethe casecase ofof mercurymercury concentrationconcentration is is computed. computed. This This is is presented presented in in Figure Figure 12 12..

Figure 12. Calibration curve for Hg2+ at diﬀerent concentrations. Figure 12. Calibration curve for Hg2+ at different concentrations. In addition to its good performance, the total cost of the device deserves special attention since it is lowerIn thanaddition 86 USD. to its Cost good discriminated performance, by the components total cost of is the presented device deserves in Table6 .special attention since it is lower than 86 USD. Cost discriminated by components is presented in Table 6.

Table 6. Total cost of the developed spectrophotometer.

Component Cost (USD) Light source 2 Stepper motor 14 Arduino UNO 21 Stepper driver 4 Light detectors 10 Black Box 20 Additional elements 15 Total 86

4. Conclusions

Sensors 2020, 20, x; doi: FOR PEER REVIEW www.mdpi.com/journal/sensors Sensors 2020, 20, 906 13 of 16

Table 6. Total cost of the developed spectrophotometer.

Component Cost (USD) Light source 2 Stepper motor 14 Arduino UNO 21 Stepper driver 4 Light detectors 10 Black Box 20 Additional elements 15 Total 86

4. Conclusions A portable low-cost spectrophotometer was developed and described in detail in this paper. The use of the proposed device has been focused on but is not limited to detection of mercuric ions Hg2+ in an aqueous medium. A particular feature to highlight of the proposed device and its test method is the use of a chemosensor which allowed the determination of mercuric ions in micromolar concentrations by means of a desulphurization reaction which was characterized by spectrophotometric methods and through 13C NMR spectroscopy. When evaluating the developed device, only slight diﬀerences were obtained between the results of the proposed device and the equivalent equipment used in a traditional laboratory. Future eﬀorts will concentrate on extending the possible application of the device to analysis of quality in fuels, determination of plasma glucose, color measurement, and even forensic applications. This work demonstrates how interdisciplinary interaction between electronics engineering and basic sciences can provide solutions facilitating a wide research and academic community having access to specialized test like this.

Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8220/20/3/906/s1, Figure S1: (a) 1W used white LED. (b) 5◦ collimator lens, Figure S2: a4988 driver in microstepping 1 configuration, Figure S3: H NMR (DMSO-d6) spectrum of 7-(diethylamino)-2H-chromen-2-one (2), Figure S4: 13 1 C NMR (DMSO-d6) spectrum of 7-(diethylamino)-2H-chromen-2-one (2), Figure S5: H NMR (DMSO-d6) 13 spectrum of 7-(diethylamino)-2H-chromene-2-thione (3), Figure S6: C NMR (DMSO-d6) spectrum of 7-(diethylamino)-2H-chromene-2-thione (3), Figure S7: HRMS of 7-(dietilamino)-2H-cromeno-2-tiona (3), Figure S8: UV-Vis and emission spectra of 3, Figure S9: (A) UV-Vis spectra of 3 at different concentration; (B) Plot of absorbance of chemosensor 3 against its concentration from 3 to 30 µM, Figure S10: (A) UV-Vis spectra of 3 at different concentration; (B) Plot of absorbance of chemosensor 3 against its concentration from 3 to 30 µM, 13 Figure S11: C NMR (DMSO-d6) spectrum of chemosensor 3 alone (A) and in the presence of mercuric ions (B), Scheme S1: Synthetic route to 7-(diethylamino)-2-chromene-2-thione (3). Author Contributions: Conceptualization, D.G.-M., O.L.-S. and O.G.-B.; Formal analysis, D.G.-M., M.F.-E., A.D.-N., A.V., O.L.-S. and O.G.-B. Funding acquisition, O.G.-B. and O.L.-S. Investigation, D.G.-M., M.F.-E., A.D.-N., A.V., O.L.-S. and O.G.-B.; Methodology, D.G.-M., O.L.-S. and O.G.-B.; Project administration, O.G.-B.; Validation, D.G.-M., M.F.-E., A.D.-N., A.V., O.L.-S. and O.G.-B.; Writing—Original Draft, D.G.-M.; Writing—Review and Editing, D.G.-M., O.L.-S. and O.G.-B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) under grant number 130774559056. Acknowledgments: This work was supported by COLCIENCIAS Grant #130774559056-Colombia. Conflicts of Interest: The authors declare that there is no conflict of interest.

References

1. Thomas, O.; Burgees, C. UV-Visible Spectrophotometry of Water and Wastewater, 2nd ed.; Elsevier Science: London, UK, 2017; p. 2. 2. Cazes, J. Analytical Instrumentation Handbook, 3rd ed.; CRC Press: Boca Raton, FL, USA, 2004; p. 127. 3. Perkampus, H.-H. UV-VIS Spectroscopy and Its Applications; Springer Science and Business Media LLC: Berlin, Germany, 1992; p. 12. Sensors 2020, 20, 906 14 of 16

4. Worsfold, P.; Zagatto, E. Spectrophotometry—Overview. In Encyclopedia of Analytical Science, 3rd ed.; Worsfold, P., Poole, C., Townshend, A., Miró, M., Eds.; Academic Press: Oxford, UK, 2019; pp. 244–248. 5. Noui, L.; Hill, J.; Keay, P.J.; Wang, R.Y.; Smith, T.; Yeung, K.; Habib, G.; Hoare, M. Development of a high resolution UV spectrophotometer for at-line monitoring of bioprocesses. Chem. Eng. Process. Process. Intensif. 2002, 41, 107–114. [CrossRef] 6. Gonzaga, F.B.; Pasquini, C. A low cost short wave near infrared spectrophotometer: Application for determination of quality parameters of diesel fuel. Anal. Chim. Acta 2010, 670, 92–97. [CrossRef][PubMed] 7. O’Toole, M.; Diamond, D. Absorbance Based Light Emitting Diode Optical Sensors and Sensing Devices. Sensors 2008, 8, 2453–2479. [CrossRef][PubMed] 8. Chuen, W.; Khoo, B.; Abdullah, A.F. A simple, low-cost and portable LED-based multi-wavelentgth light source for forensic application. Proc. Int. Soc. Opt. Photonics (SPIE) 2012, 8560, 18–27. 9. Chaianantakul, N.; Wutthi, K.; Kamput, N.; Pramanpol, N.; Janphuang, P.; Pummara, W.; Phimon, K.; Phatthanakun, R. Development of mini-spectrophotometer for determination of plasma glucose. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2018, 204, 670–676. [CrossRef] 10. Yeh, T.S.; Tseng, S.S. A Low Cost LED Based Spectrometer. J. Chin. Chem. Soc. 2013, 53, 1067–1072. [CrossRef] 11. Hardie, K.; Agne, S.; Kuntz, K.B.; Jennewein, T. Inexpensive LED-based spectrophotometer for analyzing optical coatings. Instrum. Detect. 2016, 1–19. 12. Masi, A.J.; Sesselmann, M.; Rodrigues, D.L. A Compact System for Measurement of Absorbance of Light. In Proceedings of the Society for Experimental Mechanics Series; Springer Science and Business Media LLC: Berlin, Germany, 2011; Volume 6, pp. 773–780. 13. Visconti, P.; Lay-Ekuakille, A.; Primiceri, P.; Ciccarese, G.; De Fazio, R. Hardware Design and Software Development for a White LED-Based Experimental Spectrophotometer Managed by a PIC-Based Control System. IEEE Sens. J. 2017, 17, 2507–2515. [CrossRef] 14. Albert, D.R.; Todt, M.A.; Davis, H.F. A Low-Cost Quantitative Absorption Spectrophotometer. J. Chem. Educ. 2012, 89, 1432–1435. [CrossRef] 15. Alam, A.E.; Islam, R.; Faria, I.J. Development and validation of a low-cost visible light spectrophotometer. In Proceedings of the 2017 4th International Conference on Advances in Electrical Engineering (ICAEE), Dhaka, Bangladesh, 28–30 September 2017; pp. 653–657. 16. Kittipanyangam, S.; Abe, K.; Eguchi, K. Design of a measurement device explaining the relationship between the concentration of solution and the light absorbance for chemical education. In Proceedings of the 2016 13th International Conference on Electrical Engineering/Electronics, Computer, Telecommunications and Information Technology (ECTI-CON), Chiang Mai, Thailand, 28 June–1 July 2016; pp. 1–6. 17. Liu, S.-M. The Development of a Portable Spectrophotometer for Noncontact Color Measurement. IEEE Trans. Instrum. Meas. 2004, 53, 155–162. [CrossRef] 18. Bano, S.; Altaf, T.; Akbar, S. Microcontrolled based spectrophotometer using compact disc as diffraction grid. Asia Commun. Photonics Conf. Exhib. 2010, 332–336. 19. Veras, G.; Silva, E.C.; Lyra, W.S.; Soares, S.F.C.; Guerreiro, T.B.; Santos, S.R.B.; Neto, J.G.V. A portable, inexpensive and microcontrolled spectrophotometer based on white LED as light source and CD media as diffraction grid. Talanta 2009, 77, 1155–1159. [CrossRef][PubMed] 20. Zhang, X.; Fang, Y.; Zhao, Y. A Portable Spectrophotometer for Water Quality Analysis. Sens. Transducers 2013, 148, 47–51. 21. Chen, P.-J.; Wang, H.-T.; Lin, L.-Y.; Hsu, B.-D.; Liu, D.-R.; Hwang, C.-H.; Wu, W.-H. A Practical Portable Photometer using LEDs as Inspection light source. In Proceedings of the 2017 IEEE International Instrumentation and Measurement Technology Conference (I2MTC), Turin, Italy, 22–25 May 2017; pp. 1–6. 22. Cary 60 UV-Vis Spectrophotometer. Available online: https://www.agilent.com/en/products/uv-vis-uv-vis- nir/uv-vis-uv-vis-nir-systems/cary-60-uv-vis (accessed on 12 June 2018). 23. DR-1900 Portable Spectrophotometer. Available online: https://www.hach.com/dr1900-portable- spectrophotometer/product?id=18915675456 (accessed on 12 June 2018). 24. García-Beltrán, O.; Mena, N.; Berríos, T.A.; Castro, E.A.; Cassels, B.K.; Núñez, M.T.; Aliaga, M.E. A selective fluorescent probe for the detection of mercury (II) in aqueous media and its applications in living cells. Tetrahedron Lett. 2012, 53, 6598–6601. [CrossRef] Sensors 2020, 20, 906 15 of 16

25. Cordy, P.; Veiga, M.M.; Salih, I.; Al-Saadi, S.; Console, S.; Garcia, O.; Mesa, L.A.; Velásquez-López, P.C.; Roeser, M. Mercury contamination from artisanal gold mining in Antioquia, Colombia: The world’s highest per capita mercury pollution. Sci. Total. Environ. 2011, 410, 154–160. [CrossRef][PubMed] 26. Palacios-Torres, Y.; Caballero-Gallardo, K.; Olivero-Verbel, J. Mercury pollution by gold mining in a global biodiversity hotspot, the Choco biogeographic region, Colombia. Chemosphere 2018, 193, 421–430. [CrossRef] [PubMed] 27. García-Beltrán, O.; Rodriguez, A.; Trujillo, A.; Cañete, A.; Aguirre, P.; Gallego-Quintero, S.; Núñez, M.T.; Aliaga, M.E. Synthesis and characterization of a novel fluorescent and colorimetric probe for the detection of mercury (II) even in the presence of relevant biothiols. Tetrahedron Lett. 2015, 56, 5761–5766. [CrossRef] 28. Wang, Y.; Gao, M.; Liao, C.; Yu, F.; Chen, L. A sulfydryl-based near-infrared ratiometic fluorescent probe for assessment of acute/chronic mercury exposure via associated determination of superoxide anion and mercury ion in cells and in vivo. Sens. Actuators B Chem. 2019, 301, 127038. [CrossRef] 29. He, Q.; Zhang, Q.; Cao, W.; Yin, T.; Zhao, S.; Yin, X.; Zhao, H.; Tao, W. Detecting trace of mercury ions in water using photoacoustic method enhanced by gold nanospheres. Microchem. J. 2019, 150, 104058. [CrossRef] 30. Korbas, M.; O’Donoghue, J.L.; Watson, G.E.; Pickering, I.J.; Singh, S.P.; Myers, G.J.; Clarkson, T.W.; George, G.N. The Chemical Nature of Mercury in Human Brain Following Poisoning or Environmental Exposure. ACS Chem. Neurosci. 2010, 1, 810–818. [CrossRef] 31. Zhou, Y.; Vaidya, V.S.; Brown, R.P.; Zhang, J.; Rosenzweig, B.A.; Thompson, K.L.; Miller, T.J.; Bonventre, J.V.; Goering, P.L. Comparison of kidney injury molecule-1 and other nephrotoxicity biomarkers in urine and kidney following acute exposure to gentamicin, mercury, and chromium. Toxicol. Sci. 2007, 101, 159–170. [CrossRef][PubMed] 32. Milioni, A.L.V.; Nagy, B.V.; Moura, A.L.A.; Zachi, E.C.; Barboni, M.T.; Ventura, D.F. Neurotoxic impact of mercury on the central nervous system evaluated by neuropsychological tests and on the autonomic nervous system evaluated by dynamic pupillometry. NeuroToxicology 2017, 59, 263–269. [CrossRef][PubMed] 33. Das, K.; Siebert, U.; Gillet, A.; Dupont, A.; Di-Poï, C.; Fonfara, S.; Mazzucchelli, G.; De Pauw, E.; De Pauw-Gillet, M.-C. Mercury immune toxicity in harbour seals: Links to in vitro toxicity. Environ. Health 2008, 7, 52. [CrossRef][PubMed] 34. Tan, S.W.; Meiller, J.C.; Mahaffey, K.R. The endocrine effects of mercury in humans and wildlife. Crit. Rev. Toxicol. 2009, 39, 228–269. [CrossRef][PubMed] 35. Aliaga, M.; Gazitua, M.; Rojas-Bolaños, A.; Fuentes-Estrada, M.; Durango, D.; García-Beltrán, O. A selective thioxothiazolidin-coumarin probe for Hg2+ based on its desulfurization reaction. Exploring its potential for live cell imaging. Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 2020, 224, 117372. [CrossRef][PubMed] 36. Hanna, C.P.; Tyson, J.F.; McIntosh, S. Determination of total mercury in waters and urine by flow injection atomic absorption spectrometry procedures involving on- and off-line oxidation of organomercury species. Anal. Chem. 1993, 65, 653–656. [CrossRef] 37. Darbha, G.K.; Singh, A.K.; Rai, U.S.; Yu, E.; Yu, H.; Ray, P.C. Selective Detection of Mercury (II) Ion Using Nonlinear Optical Properties of Gold Nanoparticles. J. Am. Chem. Soc. 2008, 130, 8038–8043. [CrossRef] 38. Leermakers, M.; Baeyens, W.; Quevauviller, P.; Horvat, M. Mercury in environmental samples: Speciation, artifacts and validation. TrAC Trends Anal. Chem. 2005, 24, 383–393. [CrossRef] 39. Zhang, Y.; Chen, H.; Chen, D.; Wu, D.; Chen, Z.; Zhang, J.; Chen, X.; Liu, S.; Yin, J. A colorimetric and ratiometric fluorescent probe for mercury (II) in lysosome. Sens. Actuators B Chem. 2016, 224, 907–914. [CrossRef] 40. Zhang, W.-B.; Sun, C.-X.; Yang, X.-A. Magnetic solid-phase extraction combined with in situ slurry cold vapor generation atomic fluorescence spectrometry for preconcentration and determination of ultratrace mercury. Anal. Methods 2014, 6, 2876–2882. [CrossRef] 41. Wang, L.; Zhou, J.-B.; Wang, X.; Wang, Z.-H.; Zhao, R.-S. Simultaneous determination of copper, cobalt, and mercury ions in water samples by solid-phase extraction using carbon nanotube sponges as adsorbent after chelating with sodium diethyldithiocarbamate prior to high performance liquid chromatograph. Anal. Bioanal. Chem. 2016, 408, 4445–4453. [CrossRef][PubMed] 42. Wang, N.; Lin, M.; Dai, H.; Ma, H. Functionalized gold nanoparticles/reduced graphene oxide nanocomposites for ultrasensitive electrochemical sensing of mercury ions based on thymineemercuryethymine structure. Biosens. Bioelectron. 2016, 79, 320–326. [CrossRef][PubMed] Sensors 2020, 20, 906 16 of 16

43. Wang, Y.; Jiang, L.; Leng, Q.; Wu, Y.; He, X.; Wang, K. Electrochemical sensor for glutathione detection based on mercury ion triggered hybridization chain reaction signal ampliﬁcation. Biosens. Bioelectron. 2016, 77, 914–920. [CrossRef][PubMed] 44. Bui, M.-P.N.; Brockgreitens, J.; Ahmed, S.; Abbas, A. Dual detection of nitrate and mercury in water using disposable electrochemical sensors. Biosens. Bioelectron. 2016, 85, 280–286. [CrossRef][PubMed] 45. Aliaga, M.E.; García-Río, L.; Pessêgo, M.; Montecinos, R.; Fuentealba, D.; Uribe, I.; Martín-Pastor, M.; García-Beltrán, O. Host–guest interaction of coumarin-derivative dyes and cucurbit[7]uril: Leading to the formation of supramolecular ternary complexes with mercuric ions. New J. Chem. 2015, 39, 3084–3092. [CrossRef] 46. Qin, S.; Chen, B.; Huang, J.; Han, Y. A Thiocoumarin-Based Colorimetric and Ratiometric Fluorescent Probe for Hg2+ in Aqueous Solution and Its Application in Live-Cell Imaging. New J. Chem. 2018, 42, 12766–12772. [CrossRef] 47. Bui, D.A.; Hauser, P.C. Analytical devices based on light-emitting diodes—A review of the state-of-the-art. Anal. Chim. Acta 2015, 853, 46–58. [CrossRef] 48. Shivok, T. Diﬀraction Gratings; US Naval Academy Annapolis: Annapolis, MD, USA, 2014. 49. Passos, M.L.; Saraiva, M.L.M. Detection in UV-visible spectrophotometry: Detectors, detection systems, and detection strategies. Measurement 2019, 135, 896–904. [CrossRef] 50. Gonzalez-Morales, D.F.; Lopez-Santos, O.; Garcia-Beltran, O. Low-Cost Spectrophotometer for In-Situ Detection of Mercury in Water. In Proceedings of the 2018 IEEE SENSORS, New Delhi, India, 28–31 October 2018; pp. 1–4.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).