Easy Diagnosis of Jaundice: A Smartphone-based Nanosensor

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Easy Diagnosis of Jaundice: A Smartphone-based Nanosensor Bioplatform using Photoluminescent Bacterial Nanopaper for Point-of-Care Diagnosis of Hyperbilirubinemia Raziyeh Sadat Tabatabaee, Hamed Golmohammadi, and Seyyed Hamid Ahmadi ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.9b00275 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Easy Diagnosis of Jaundice: A Smartphone-based Nanosensor Bioplatform using Photoluminescent Bacterial Nanopaper for Point-ofCare Diagnosis of Hyperbilirubinemia Raziyeh Sadat Tabatabaee, Hamed Golmohammadi*, Seyyed Hamid Ahmadi* Chemistry and Chemical Engineering Research Center of Iran, 14335-186, Tehran, Iran

* Corresponding author: Hamed Golmohammadi, Seyyed Hamid Ahmadi Email : [email protected], [email protected] [email protected],[email protected] Tel: +98 21 44787720-40 (Ext: 1103)

KEYWORDS: Photoluminescence, Jaundice; Point-of-Care diagnosis; Smartphone-based sensors; Bacterial nanopaper. ABSTRACT: One of the concerns of parents in the first days of their baby's birth is the baby's risk of jaundice/hyperbilirubinemia. This is because more than 60 percent of babies are born with jaundice that, if not timely diagnosed and subsequently treated, can lead to serious damage to their health. On the other hand, despite recent progresses in sensors technology for clinical applications, the development of easy-to-use, cost-effective, sensitive, specific and portable diagnostic devices, which use non-toxic and biodegradable materials in their design and fabrication, are still demanded. Herein we present an easy-to-use, cost-effective, selective, non-toxic and disposable photoluminescent nanopaper-based assay kit with a smartphone readout for easy diagnosis of neonatal jaundice through visual determination of Bilirubin (BR) in infants’ blood samples. The developed BR assay kit comprises highly photoluminescent carbon dots (CDs) sensing probes embedded in a bacterial cellulose (BC) nanopaper substrate (CDBN). The photoluminescence (PL) of the developed BR sensor is quenched in the presence of BR as a PL quencher and then selectively recovered upon blue light (λ=470 nm) exposure, due to converting the unconjugated BR to the colourless oxidation products(non-PL quencher) through BR photoisomerization and photooxidation, that subsequently leads to selective PL enhancing of CDBN. The recovered PL intensity of the developed BR assay kit, which was monitored by integrated smartphone’s camera, was linearly proportional to the concentration of BR in the range of 2-20 mg dL-1. The feasibility of real application of the fabricated smartphone-based BR assay kit was also confirmed via comparing the results of our method with a clinical reference method for determination of BR concentration in infant’s blood samples. Taking the advantages of the nontoxicity and the extraordinary physicochemical properties of photoluminescent BC nanopaper as sensing substrate, along with those of smartphone technology, we believe that our developed smartphone-based BR assay kit, as an easy-to-use, cost-effective (~0.01 Euro per test), portable and novel sensing bioplatform, can be potentially exploited for sensitive, specific, rapid and easy BR detection and jaundice diagnosis at the point-of-care (POC) and routine clinical laboratories as well.

The birth of a baby in a family brings a sense of happiness and satisfaction, while the health of this baby also has its own concerns. One of the factors affecting neonatal health concern for parents, especially during the first days after the baby’s birth, is the baby's risk of jaundice. Nearly 60 percent of terms and 80 percent of preterm infants have jaundice on the first days of life.1-2 Neonatal jaundice/hyperbilirubinemia is a condition that the skin gets a yellow tint and the whites of eyes turn yellow, orange or brown due to the accumulation of excess (over 2–3 mg dL-1) Bilirubin (BR) in blood.3-5 BR is a yellowish compound that is produced during the destruction process of aged red blood cells in liver. The breakdown of heme is followed by production of biliverdin as the first step of catabolism and BR is created through the reduction of biliverdin with bilirubin reductase enzyme.6-7 There are two types of BR; conjugated (direct) BR, a watersoluble version that is easily excreted in urine and faeces, and a water-insoluble form of BR, called unconjugated (indirect)

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BR that cause neonatal jaundice.8-9 The normal BR level in the blood of infants is about 2 mg dL-1 and would be under 5 mg dL-1 within the first 12 hours of birth. But many infants have jaundice and their blood BR level rises above 5 mg dL-1within the first few days after birth. For the first 72 hours of birth, increasing the blood BR level more than 15 mg dL-1 has the high risk of hyperbilirubinemia effects.10-11 Untreated sever jaundice can cause deafness, cerebral palsy, acute bilirubin encephalopathy and kernicterus in neonates.12-16 Due to these serious health concerns, the accurate and timely diagnosis and consequently early treatment of jaundice are arguably crucial. Different methods are used for treatment of neonatal jaundice such as phototherapy, intravenous immunoglobulin, pharmacological treatment and exchange transfusion.17-19 Among these, phototherapy is the most common method using visible light, especially blue light tubes, for the treatment of hyperbilirubinemia in the newborns. Upon blue light exposure, the level of BR in blood and skin is decreased dramatically due to photoisomerization of unconjugated BR to the conjugated form.2, 20-22 However, before treatment, the use of an accurate diagnostic/analytical method for determination of BR level in infant’s blood sample to determine whether the baby is suffering from jaundice is necessary. Thus far, different methods have been reported for determination of BR level in biological fluids, including spectroscopic,23-25 electrochemical,26-28 liquid chromatographic methods.29-31 Although, these approaches have demonstrated good performance for BR detection, they cannot be properly considered as user-friendly methods, since most of them require high volumes of sample, toxic chemicals and also relatively expensive, large and sophisticated laboratory equipment with trained operators that will consequently restrict their applications to laboratories. Moreover, the laboratory turnaround time (TAT) of these approaches from sample collection to publishing test results is almost too long that can leads to decrease the real time estimation possibility for point of care testing (POCT) systems.32-33 There are some portable BR meters which are handheld and operate in transcutaneous settings and giving the results for POCT, but their answers are depended on the skin colour of infant, furthermore Haemoglobin is the major absorber of visible light in the dermis, especially in the regions 400–425 nm and 500–600 nm and competes the absorbance of BR, and subsequently affects the results.34-36 Moreover, transcutaneous bilirubin measurements in newborns have not the same accuracy of blood-based methods. The results of transcutaneous bilirubin measurements will be different depending on the infants body sites (e.g. forehead, sternum and interscapular region) and for the first hours of birth the transcutaneous bilirubinometers overestimate the TcB at least 2-3 mg dL-1 in comparison to TsB.37 To attenuate above complications, the development of new sensing platforms/tools for BR detection, which are efficient, sensitive, specific, affordable, easy-to-use, disposable, rapid and amenable to automation and portability and also require small volumes of sample and reagents, is still demanded. Bacterial cellulose (BC) nanopaper-based (bio)sensing platforms, as new generation of paper-based analytical devices, have recently attracted considerable interest in various (bio)sensing applications including environmental monitoring, the detection of toxic materials, food quality control and clinical/medical diagnostics, which is attributed to the fascinating and unique physicochemical characteristics of BC nanopaper. In fact, BC nanopaper, as a recent alternative to paper, is known as a continuous sheet/film of nanosized cellulose fibres that is commonly produced via “bottom-up” synthetic routes from carbon sources such as glucose by some specific nonpathogenic bacteria such as Gluconacetobacter xylinus. Apart from having the advantageous properties of cellulosic papers including low cost, flexibility, porous matrix, printability, biocompatibility and biodegradability, BC nanopaper has optical transparency, low thermal expansion and surface roughness, high mechanical and chemical stability that make it a promising substrate/platform in (bio)sensing technology.38-42 On the other hand, up to now, measurement and monitoring of analytes has mostly relied on expensive, non-portable and sophisticated devices, which limit their usage to laboratories with trained operators. A new generation of low cost, portable and easy-to-use analytical devices, which require only a simple scanner, have been introduced to overcome the mentioned problems of the conventional analytical devices. Recently, the use of smartphone cameras, as the most common and easy-touse scanners with high sensitivity for light and colour changes, has been proven to be powerful analytical tools to digitize the images of the colorimetric measurements. The coupling of nanopaper-based sensors to smartphones can greatly enhance the applications of these (bio)sensing platforms and open up opportunities towards the development of costeffective, portable and user-friendly monitoring devices.43-46 Herein we report an easy-to-use, cost-effective, non-toxic, disposable and portable assay kit, which has been coupled with a smartphone as colour signal reader, for sensitive, specific, rapid and easy diagnosis of neonatal jaundice via visual determination of BR level in infants’ blood samples. The highly photoluminescent CDs embedded in BC nanopaper (CDBN) were used as sensing elements in the developed BR assay kit. The PL of the developed CDBN is quenched upon addition of BR and then selectively recovered under blue light irradiation (λ=470 nm). The recovered PL of the developed BR sensor, which is monitored by integrated smartphone’s camera, is utilized as analytical signal for BR detection. Two UV LED lamps (λ=365 nm) that excite the CDBN, and two blue LED lamps (λ=470 nm) for photoisomerization of BR, were placed within a homemade dark chamber. Each test zone is processed, one by one, in order to reach the LEDs area. A mobile phone was utilized as digital colour imaging capture and also energy source that connected to the LEDs through an USB port. The details of fabrication and characterization of CDBN platform, fabrication and set up of smartphone-based BR sensor and investigation of affecting parameters on analytical performance of developed BR assay kit are described. The analytical characteristics of the developed smartphone-based assay kit for quantitative determination of BR were also evaluated for selectivity, sensitivity, linearity and analysing the real samples.


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EXPERIMENTAL SECTION Materials Milli-Q grade water, with a resistivity of 18.2 MΩ, was used in all experiments, and all chemicals were of analytical grade. Citric acid and ethylene diamine (EDA) were purchased from Merck. Bilirubin was supplied from Man Co., Iran. Total Bilirubin Assay Kit from Audit Diagnostics; Ireland, was used as reference method for clinical determination of BR concentration. A wet form of BC nanopaper (350 mm long, 250 mm width and 3 mm thickness) containing 1 wt% solid content/ 99% deionized water, was supplied from Nano Novin Polymer Co., Iran. Blood separation membrane, MF1, was purchased from GE Healthcare; United States, in order to filter red blood cells from Whole Blood samples without centrifugation. Equipment The images were taken by Samsung Galaxy A5-2016, in manual mode, auto focus on shutter speed 1/127, ISO 40, 16 MP. Blue light power LED from Epileds, Taiwan with λ=470 nm, light intensity 23-60 Lm, 3.0 volt and maximum 350 mA electric current and a black-light blue UV LED, from Epileds, Taiwan with λ=365 nm, 3.3 volt and maximum 700 mA electric current, were used for illumination. The PL spectra were carried out by Jasco Model FP-6500 Spectro-Fluorometer. Transmission electron microscopic (TEM) image and dynamic light scattering (DLS) analysis of CDs were performed by Zeiss – EM900 high-resolution transmission electron microscope operating at 80 KV and a particle size analyzer (Scatteroscope I; Qudix, Inc., South Korea), respectively. An office laser printer (HP LaserJet P1102) was used to create the hydrophilic test zones with the hydrophobic barriers via printing the toner layer on the dried BC nanopaper film. Confocal microscopy was performed through a Leica TCS SPE. Synthesis of photoluminescent CDs Photoluminescent CDs were synthesized through a hydrothermal method.1 500 µL EDA was added to 500 mg citric acid and the mixture was diluted with 10 mL water and then transferred to a Teflon-lined stainless-steel autoclave and heated for 5 hours at 200 °C.47 The TEM image, DLS analysis and PL spectra of the synthesized CDs have been depicted in Figure S1 and S2 respectively. BR assay kit fabrication and set up The wet BC nanopaper sheets were dewatered by placing them between two normal filter papers, which were sandwiched between two glass plates (5 mm in diameter) and fold back clips. This set up was then dried in an oven for 12 hours at 100 °C. Following that, the dried BC nanopaper film was separated from one of blotting papers.39 To create the patterned layouts (multiwall circles), an office LaserJet printer was used. The desired pattern was first created in Microsoft Word 2016 as transparent circles with 5mm diameter on a black background. The circular hydrophilic test zones with hydrophobic walls were directly printed onto the dried BC nanopaper film. Finally, after printing the desired pattern, the second blotting paper was separated from the printed BC nanopaper film. To fabricate the CDBN sensing platform, 5 μL volumes of the pre-prepared CDs were dropped in test zones of the fabricated BC nanopaper platforms. The drop-casted solutions were then dried at room temperature for 30 minutes and defined as the sensing probes. A 3D-printed dark chamber with defined places for LED lamps (two UV LED lamps (λ=365 nm) that excite the CDBN, and two blue LED lamps (λ=470 nm) for photoisomerization of BR) and smartphone camera, which can be well adapted with mobile phone, was fabricated. The assembled sensing setup is depicted in Scheme 1, Figures S3 and S4. The CDBN was first placed within the dark chamber through a strip hole to access the defined place for UV LED (λ= 365 nm) and blue light LED (λ=470 nm). LEDs were powered by smartphone using an USB port. The change in the PL intensity of CDs, which was captured by integrated smartphone’s camera, was quantified in relation to the analyte concentration using our self-developed application. Recommended process for determination of BR using the fabricated CDBN platforms The experimental process for determination of BR by the fabricated smartphone-based BR assay kit was as followed: 5 µL of the BR standard solutions were added to each of the hydrophilic test zones of the fabricated CDBN platform. The pictures of the test zones were taken with the smartphone’s camera adapted to the fabricated dark chamber under the irradiation of UV lamps (λ= 365 nm) embedded in the fabricated chamber. The test zones were then lightened up with blue light lamps (λ= 470 nm) embedded in the fabricated dark chamber for 5 min and the above process was run again. A blank was also prepared in the same procedure, except the addition of BR. The taken images were processed and analysed using our selfdeveloped mobile application, to measure the mean colour intensity (in grey mode) of each test zones and subsequently to determine the related BR concentrations via the detection algorithm (Scheme 2A, Figure S5). Real sample analysis To assess the clinical capability of the developed smartphone-based BR assay kit, the human blood samples were obtained from neonates between the ages of 1 to 7 days by collecting one drop blood specimen via blood-sampling device


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and put on the plasma separation membrane in order to separate red blood cells from samples and then subjected to our recommended procedure for the determination of BR concentration and also for further validation assayed by using Audit Total Bilirubin Assay Kit as the clinical reference method for BR level detection.

RESULTS AND DISCUSSION Sensing strategy The aim of current research was to develop an easy-to-use, cost-effective, safe and portable sensing device _as a smartphone-based assay kit_ that can be utilized for easy, selective and rapid detection of BR and jaundice diagnosis at the POC and clinical laboratories. The highly photoluminescent CDs embedded in BC nanopaper (CDBN) were utilized as sensing probe in the developed BR sensing bioplatform. The PL of the developed BR sensor is quenched in the presence of BR as a PL quencher and then selectively recovered upon blue light (λ=470 nm) exposure. Figure 1A(a-c) illustrates the PL spectra of CDs solution in the absence and presence of 10 mg dL-1 of BR, before and after exposure to blue light irradiation (λ=470 nm). As shown in this figure, the PL emission of CDs with a characteristic peak at λ=465 nm is decreased in the presence of BR, and then recovered again under blue light irradiation. The UV-vis absorption spectra of BR (10 mg dL-1), before and after exposure to blue light irradiation, are also shown in Figure 1A(d and e). The PL quenching mechanism of the fabricated CDBN upon addition of BR can be attributed to the fluorescence resonance energy transfer (FRET)and the inner filter effect (IFE) of BR towards CDs, which is originated from the overlap between UV-vis absorption spectrum of BR (Figure 1A(d)) and PL emission of CDBN (Figure 1A(a)).48 The sensing strategy for BR detection, as schematically depicted in Scheme 2A, is based on selective recovering the quenched PL of the mixture of CDs-BR, upon blue light (λ=470 nm) exposure, which is commonly used as a routine method in photography’s treatment of jaundice in newborns, due to photoisomerization and photooxidation of BR and converting the unconjugated BR to the colourless products (non-PL quencher)that subsequently led to selective PL increasing. There are several reports on natural photoisomerization of BR upon blue light irradiation (λ=470-490 nm). In unconjugated BR structure, the bridging exocyclic double bond in each of the two dipyrrinone has a Z configuration as the most stable form. The unconjugated isomer is a water insoluble form of BR that is stabilized via intramolecular H-bonding. In the presence of blue light, the configurational isomerization is occurred and lumirubins are formed.49-52 Since BR is an oxygen-sensitive molecule, in the presence of dissolved oxygen in the samples, the photooxidation process occurs following the photoisomerization and subsequently the polar, colourless and water-soluble oxidation products are formed (Scheme 2B).53-56 Through this photoreaction, the dipyrrinone chromophore gets free from intermolecular hydrogen bonds and the molecule breaks into smaller pieces.51-52 Although this photoisomerization reaction is occurred slowly in the body, at the microfluidic level and thin layers it can change faster. To elucidate the observed BR sensing characteristics in the fabricated CDBN platform, we performed confocal microscopy analysis. Figure 1B illustrates the confocal microscopy images of the bare BC nanopaper and the fabricated CDBNs in the presence of different BR concentrations before and after exposure to blue light, which clearly confirms the PL changes caused by the BR photoisomerization process and consequently the recommended sensing strategy for BR detection using the developed assay kit. Storage time and the stability of the fabricated CDBNs Since the stability and storage time of sensing elements embedded in sensor substrate are of great importance on the efficiency, reproducibility and applicability of the developed BR assay kit, the PL stability and the sensor storage of the fabricated CDBNs were evaluated upon time by storing them at room temperature and recording the corresponding PL. As can be seen in Figure S6, the PL intensities of the fabricated CDBNs remained constant even after 3 months, which indicates the long storage timeand the stabilityof the fabricated CDBNs for practical applications. Optimization of effective variables on the performance of the developed smartphone-based BR assay kit To provide the optimum conditions and promote the efficiency of the developed smartphone-based BR assay kit, the effect of main parameters, including the ratio of CDs dilution and the time duration of blue light illumination were evaluated. To assess the influence of the ratio of CDs dilution, the BR monitoring procedure using the developed assay kit was examined in various ratio of CDs in the range of 0.02 to 1.0. From the experimental results shown in Figure S7 A, it was found that the maximum of PL signal was obtained when the CDs solution was diluted 10 times. Hence, the ratio of CDs dilution about 0.1 was chosen for further experiments. During the course of this investigation, it was found that BR monitoring using the developed assay kit is dependent on the blue light irradiation time. BR sample with concentration of 15 mg dL-1 was added to the test zone and illuminated 10 min with imaging every minute. As shown in Figure S7 C, as the blue light irradiation time was prolonged, the analytical signal changed quickly and eventually was constant after 5 min. As a result, duration of 5 min was regarded as the optimal blue light illumination time for BR detection. Analytical performance of the developed smartphone-based BR assay kit Under the optimal experimental conditions, the analytical characteristics of the developed smartphone-based BR assay kit were evaluated. The calibration curve was linear in the range of 2-20 mg dL-1 of BR with a correlation coefficient (r) of


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0.9950. The linear regression equation for BR was ΔS = 2.772C + 5.664, where ΔS is the analytical signal and C is the BR concentration in mg dL-1 (Figure 2). The limit of detection (S/N= 3) for BR was found to be 0.19 mg dL-1. The relative standard deviation for seven replicate measurements of 10 mg dL-1 of BR was 1.87 %, which verifies the results obtained with this developed BR assay kit are reproducible. Interference study To evaluate the selectivity of the fabricated sensing bioplatform for the BR determination, the competitive experiments were carried out at 10 mg dL-1 of BR in the presence of some (bio)chemicals probably existing in blood: Glucose (125 mg dL1), Total Protein (8000 mg dL-1), Albumin (5500 mg dL-1), Uric acid (7 mg dL-1), Creatinine (0.6 mg L-1), Urea (18 mg dL-1), Cyanocobalamin (0.1 mg dL-1), L-ascorbic acid (1.5 mg dL-1), L-Phenylalanine (1 mg dL-1), Na+ (330 mg dL-1), K+ (20 mg dL1), Mg2+ (2 mg dL-1), Ca2+ (10.2 mg dL-1), Zn2+ (130 µg dL-1), Cl- (340 mg dL-1), HCO - (140 mg dL-1), according to the 3 recommended procedure. Figure 3A indicates the good selectivity of the developed smartphone based-assay kit toward BR. Although there are many quenchers in blood, only BR responds to the blue light and rearranged due to photoisomerization, hence the analytical response of the CDBNs was little affected (less than ±5% from the analytical response of BR) in the presence of other interference species. Application of the developed smartphone-based assay kit toward BR sensing in infants’ blood samples The clinical applicability of the developed smartphone-based BR assay kit was validated by analysing the blood samples of 10infants and comparing the results of our developed sensor with Audit Total Bilirubin Assay Kit as a clinical reference method for BR level detection in blood samples. One drop blood specimen was taken via blood-sampling device and put on the plasma separation membrane in order to separate red blood cells from samples. The final solution was then added to CDBNs and analysed. Figure 3 shows the results of both methods that have been plotted against each other to compare the methods. The regression coefficient of this comparison (r=0.9985) reveals the results and performance of our developed smartphone-based BR assay kit is comparable to reference methods that are routinely used for determination of BR level in blood samples and jaundice diagnosis in clinical laboratories. The obtained results indicate the usability of the developed smartphone-based assay kit for BR level detection at the POC and also clinical laboratories. CONCLUSIONS In conclusion, we have developed for the first time an easy-to-use, cost-effective, non-toxic and portable smartphone-based diagnostic bioplatform capable of neonatal jaundice diagnosis through visual monitoring of BR in infants’ blood samples. The developed BR assay kit comprises highly photoluminescent CDs-decorated BC nanopaper as sensing probe, which has been coupled with smartphone technology. The sensing strategy for BR detection was based on selective recovering the quenched PL of the mixture of CDs-BR upon blue light (λ=470 nm) exposure, due to photoisomerization of BR. Indeed, although there are many (bio)chemical compounds in human blood that may lead to PL quenching of the CDs but only BR can be photo-isomerized under blue light irradiation and consequently selectively recover the quenched PL of CDs. The comparison of the results obtained from our developed sensor with a clinical reference method for determination of BR concentration in blood samples reveals the clinical efficiency and usability of the developed smartphone-based assay kit. It is noteworthy that the developed assay kit can also be considered as a green diagnostic device due to utilizing the CDs and BC nanopaper as non-toxic, inexpensive and green materials, the small required blood volume (one drop) with minimum TAT and not requiring sophisticated instrumentation for BR sensing. Considering the advantages of the non-toxicity and remarkable physicochemical features of CDs-decorated BC nanopaper as sensing substrate, in combination with ubiquitous availability of smartphones, we are confident that our developed-based diagnostic bioplatform, as an easy-to-use, costeffective (~0.01 Euro per test) (See Table S1) and portable sensing bioplatform, can be potentially exploited for specific, straightforward, rapid and easy sensing of BR and jaundice diagnosis at the POC and also routine clinical laboratories.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Characterization of Photoluminescent CDs; images and illustration of 3D-printed sensing setup and our self-developed mobile application, diagrams of time, stability and effective variables on performance of sensing platform, table of estimated costs.

ACKNOWLEDGMENT Financial support from Chemistry & Chemical Engineering Research Centre of Iran (Tehran, Iran) and the Nano Match program of Iran Nanotechnology Initiative Council (INIC) are gratefully acknowledged.


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Notes The authors have filed a provisional patent application on the technology described in this paper.

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Journal of Electroanalytical Chemistry 2018,818, 124-130. 29. Ma, G.; Lin, J.; Cai, W.; Tan, B.; Xiang, X.; Zhang, Y.; Zhang, P., Simultaneous determination of bilirubin and its glucuronides in liver microsomes and recombinant UGT1A1 enzyme incubation systems by HPLC method and its application to bilirubin glucuronidation studies. Journal of pharmaceutical and biomedical analysis 2014,92, 149-159. 30. Martelanc, M.; Žiberna, L.; Passamonti, S.; Franko, M., Direct determination of free bilirubin in serum at sub-nanomolar levels. Analytica chimica acta 2014,809, 174-182. 31. Martelanc, M.; Žiberna, L.; Passamonti, S.; Franko, M., Application of high-performance liquid chromatography combined with ultrasensitive thermal lens spectrometric detection for simultaneous biliverdin and bilirubin assessment at trace levels in human serum. Talanta 2016,154, 92-98. 32. Alan B. Storrow; Chuan Zhou; Gary Gaddis; Jin H. Han; Karen Miller; David Klubert; Andy Laidig; Aronsky, D., Decreasing Lab Turnaround Time Improves Emergency Department Throughput and Decreases Emergency Medical Services Diversion: A Simulation Model. Academic Emergency Medicine 2008,15 (11), 1130-1135. 33. Price, C. P., Point of care testing: Potential for tracking disease management outcomes. Disease Management & Health Outcomes 2002,10 (12), 749-761. 34. Maisels, M. J.; Ostrea, E. M.; Touch, S.; Clune, S. E.; Cepeda, E.; Kring, E.; Gracey, K.; Jackson, C.; Talbot, D.; Huang, R., Evaluation of a new transcutaneous bilirubinometer. Pediatrics 2004,113 (6), 1628-1635. 35. Bental, Y.; Shiff, Y.; Dorsht, N.; Litig, E.; Tuval, L.; Mimouni, F., Bhutani-based nomograms for the prediction of significant hyperbilirubinaemia using transcutaneous measurements of bilirubin. Acta Paediatrica 2009,98 (12), 1902-1908. 36. Lamola, A. A.; Bhutani, V. K.; Wong, R. J.; Stevenson, D. K.; McDonagh, A. F., The effect of hematocrit on the efficacy of phototherapy for neonatal jaundice. Pediatric research 2013,74 (1), 54-60. 37. Clark, K., Are transcutaneous bilirubin measurements in newborns a suitable estimate of serum bilirubin? Evidence-Based Practice 2019,22 (3), 16-16. 38. Pourreza, N.; Golmohammadi, H.; Naghdi, T.; Yousefi, H., Green in-situ synthesized silver nanoparticles embedded in bacterial cellulose nanopaper as a bionanocomposite plasmonic sensor. Biosensors and Bioelectronics 2015,74, 353-359.


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39. Morales-Narváez, E.; Golmohammadi, H.; Naghdi, T.; Yousefi, H.; Kostiv, U.; Horák, D.; Pourreza, N.; Merkoçi, A., Nanopaper as an optical sensing platform. ACS nano 2015,9 (7), 7296-7305. 40. Heli, B.; Morales-Narváez, E.; Golmohammadi, H.; Ajji, A.; Merkoçi, A., Modulation of population density and size of silver nanoparticles embedded in bacterial cellulose via ammonia exposure: visual detection of volatile compounds in a piece of plasmonic nanopaper. Nanoscale 2016,8 (15), 7984-7991. 41. Golmohammadi, H.; Morales-Narvaez, E.; Naghdi, T.; Merkoci, A., Nanocellulose in Sensing and Biosensing. Chemistry of Materials 2017,29 (13), 5426-5446. 42. Abbasi-Moayed, S.; Golmohammadi, H.; Hormozi-Nezhad, M. R., A nanopaper-based artificial tongue: a ratiometric fluorescent sensor array on bacterial nanocellulose for chemical discrimination applications. Nanoscale 2018,10 (5), 2492-2502. 43. Lopez-Ruiz, N.; Curto, V. F.; Erenas, M. M.; Benito-Lopez, F.; Diamond, D.; Palma, A. J.; Capitan-Vallvey, L. F., Smartphone-based simultaneous pH and nitrite colorimetric determination for paper microfluidic devices. Analytical chemistry 2014,86 (19), 9554-9562. 44. Xu, Y.; Xie, X.; Duan, Y.; Wang, L.; Cheng, Z.; Cheng, J., A review of impedance measurements of whole cells. Biosensors and Bioelectronics 2016,77, 824-836. 45. Álvarez-Diduk, R.; Orozco, J.; Merkoçi, A., Paper strip-embedded graphene quantum dots: a screening device with a smartphone readout. Scientific reports 2017,7 (1), 976-984. 46. You, M.; Lin, M.; Gong, Y.; Wang, S.; Li, A.; Ji, L.; Zhao, H.; Ling, K.; Wen, T.; Huang, Y., Household fluorescent lateral flow strip platform for sensitive and quantitative prognosis of heart failure using dual-color upconversion nanoparticles. ACS nano 2017,11 (6), 6261-6270. 47. Zhu, S.; Meng, Q.; Wang, L.; Zhang, J.; Song, Y.; Jin, H.; Zhang, K.; Sun, H.; Wang, H.; Yang, B., Highly photoluminescent carbon dots for multicolor patterning, sensors, and bioimaging. Angewandte Chemie 2013,125 (14), 4045-4049. 48. Senthilkumar, T.; Asha, S., Selective and sensitive sensing of free bilirubin in human serum using water-soluble Polyfluorene as fluorescent probe. Macromolecules 2015,48 (11), 3449-3461. 49. Bruzell Roll, E.; Christensen, T., Formation of photoproducts and cytotoxicity of bilirubin irradiated with turquoise and blue phototherapy light. Acta Paediatrica 2005,94 (10), 1448-1454. 50. Zietz, B.; Gillbro, T., Initial photochemistry of bilirubin probed by femtosecond spectroscopy. The Journal of Physical Chemistry B 2007,111 (41), 11997-12003. 51. Lisenko, S.; Kugeiko, M., Method for Estimating Bilirubin Isomerization Efficiency in Phototherapy to Treat Neonatal Jaundice. Journal of Applied Spectroscopy 2014,81 (5), 834-842. 52. Mreihil, K.; Madsen, P.; Nakstad, B.; Benth, J. Š.; Ebbesen, F.; Hansen, T. W. R., Early formation of bilirubin isomers during phototherapy for neonatal jaundice: effects of single vs. double fluorescent lamps vs. photodiodes. Pediatric research 2015,78 (1), 56-62. 53. Shishido, N.; Nakayama, K.; Nakamura, M., Porphyrin-Induced Photooxidation of Conjugated Bilirubin*. Free radical research 2003,37 (10), 1061-1067. 54. Cardoso, L. C.; Savedra, R. M.; Silva, M. M.; Ferreira, G. R.; Bianchi, R. F.; Siqueira, M. F., Effect of blue light on the electronic and structural properties of Bilirubin isomers: insights into the photoisomerization and photooxidation processes. The Journal of Physical Chemistry A 2015,119 (34), 9037-9042. 55. Plavskii, V. Y.; Mostovnikov, V.; Tret’Yakova, A.; Mostovnikova, G., Sensitizing effect of Z, Z-bilirubin IXα and its photoproducts on enzymes in model solutions. Journal of Applied Spectroscopy 2008,75 (3), 407-419. 56. Ritter, M.; Seidel, R. A.; Bellstedt, P.; Schneider, B.; Bauer, M.; Görls, H.; Pohnert, G., Isolation and identification of intermediates of the oxidative bilirubin degradation. Organic letters 2016,18 (17), 4432-4435.

Scheme 1. The images of our 3D-printed sensing setup A and B, C) The captured image of sample, The image of our selfdeveloped mobile application: D) The main window, E) The image processing view.


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Scheme 2. A) The illustration of our photoluminescent nanopaper-based BR assay kit with a smartphone readout, B) The photoisomerization process of BR in the presence of blue light (λ=470 nm).


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Figure 1. A) The PL spectra of a) CDs solutions in the absence and b) presence of 10 mg dL-1 of BR before and c) after exposure to blue light for 30 minutes. The UV-vis absorption spectra of BR (10 mg dL-1) d) before and e) after exposure to blue light irradiation. B) Confocal microscopy images of bare BC nanopaper (a) before and (f) after exposure to blue light λ=470 nm for 5 min, and CDBNs after addition of 5 µL BR with concentrations of 0, 5, 10 and 15 mg dL-1 (b, c, d, e) before and (g, h, i, j) after exposure to blue light λ=470 nm for 5 min (excitation wavelength 365 nm, maximum emission 465 nm).


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Figure 2. A) Calibration plot of BR sensing using the developed smartphone-based BR assay kit in the range of 2-20 mg dL-1 of BR. The images of the fabricated CDBN platforms through increasing the concentration of BR (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mg dL-1 from left to right)observed under the UV light B) before and C) after of blue light exposure. Error bars were obtained by taking the standard deviation of three tests (n=3) in the same conditions.


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Figure 3. A) The effect of some coexisting (bio)chemicals on the analytical response of the fabricated CDBN toward BR sensing, B) Correlation between obtained results of BR analysis using the developed smartphone-based BR assay kit and Audit Total Bilirubin assay kit as reference method recommended for clinical uses, The application of fabricated CDBN platforms for determination of BR in infant’s blood samples C) before and D) after exposure to blue light irradiation. Error bars were obtained by taking the standard deviation of three tests (n=3) in the same conditions.


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Table of Content


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Easy Diagnosis of Jaundice: A Smartphone-based Nanosensor

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