Functionalization of Gold Nanoparticles for a Color-Based Detection of

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Functionalization of Gold Nanoparticles for a Color-Based Detection of Adenosine in a Bioassay Yoann Roupioz* Université Grenoble Alpes, CNRS, CEA, INAC-SyMMES, 38000 Grenoble, France

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S Supporting Information *

ABSTRACT: A simple bioassay was designed for the detection of adenosine, a human metabolite involved in several physiological processes, on the basis of the functionalization of gold nanoparticles with DNA oligonucleotides selectively binding adenosine. The test was possible thanks to the selective and sensitive binding of adenosine by two DNA oligonucleotides forming an aptamer. Each DNA strand was grafted on the surface of gold nanoparticles, which formed an insoluble aggregate within a few minutes in the presence of millimolar concentrations of adenosine. The experiments described are suitable for undergraduate and graduate students developing skills in handling and functionalizing nanomaterials for use in biosensors. Besides the handling of nanomaterials, this laboratory training enabled the students to familiarize themselves with the use of nucleic acids in novel techniques, for example, as efficient probes for biosensing purposes. KEYWORDS: Graduate Education/Research, Bioanalytical Chemistry, Analytical Chemistry, Nanotechnology, Nucleic Acids/DNA/RNA, Hands-On Learning/Manipulatives, Molecular Recognition, Laboratory Instruction

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INTRODUCTION With industry’s increasing interest in nanoscience-based methods, it has become mandatory to include a basic introduction to nanotechnologies in the training of undergraduate and graduate students following degree courses in chemistry. In this field, nanoparticles occupy quite a unique position as they can require competency in chemical synthesis and characterization, functionalization and conjugation of biomolecules, toxicity and biodistribution of nanomaterials, and waste disposal and control of environmental impact. This practical experience has been set-up to give the opportunity to undergraduate and graduate students to have an overview of each of these aspects as they relate to nanoparticles. Several cohorts of students, mostly graduate students, attended this practical during two runs of two-day sessions of the 2017 international NanoAndes Summer School (held in Buenos Aires, Argentina) and for the 2018 European School On Nanosciences & Nanotechnologies1 (held in Grenoble, France). Students attending the NanoAndes Summer School came mostly from Central and South America (Costa Rica, Peru, Argentina, Brazil, Colombia, Ecuador, Venezuela), while students attending the ESONN School were from European countries and India. Several groups of mostly graduate students (2 or 3 students in each group) successfully completed the entire experimental protocol. Each session lasted 4−5 h, depending on student fitness. For most students, this practical session was their first experience in handling, functionalizing, and using nanomateri© XXXX American Chemical Society and Division of Chemical Education, Inc.

als. They appreciated the instrument-free approach to explore the functional properties of nanoparticles, as well as the easy, and reversible, bioassay which they built themselves. A short introduction on molecular structure and properties of nucleic acids came before the experimental work. Use of Aptamers for the Detection of Adenosine

Adenosine is an organic intermediate involved in a variety of different biochemical pathways and is present in eukaryotic cells at nanomolar up to micromolar levels.2 Adenosine can be found in several human organs such as the nervous, cardiovascular, gastrointestinal, urogenital, respiratory, and lymphatic systems2−4 with increasing concentration in stressed conditions. For this reason, this product may be used as a metabolic stress biomarker.5 Aptamers are short DNA or RNA sequences (polymers usually less than 60 nucleotides long), which self-assemble into 3D structures exhibiting some affinity to a predetermined target.6,7 Their affinity and specificity toward a target make them interesting alternatives to antibodies in immunoassays. In 1995, Huizenga and Szostak identified a DNA aptamer sequence recognizing not only adenosine but also the adenosine triphosphate (ATP),8 which is also a major player in the regulation of cellular metabolism and biochemical cascades.9 Since then, this 27-base-long Received: January 16, 2019 Revised: March 7, 2019

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DOI: 10.1021/acs.jchemed.9b00044 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Finally, the most important pedagogical objective of this laboratory training was the design and performance assessment of a bioassay for the sensitive detection of the adenosine nucleoside. This performance assessment is a crucial step in the development of any sensor or biosensor, and this requirement informed the design by the students of alternative candidate molecular systems and dedicated controls for the assay qualification. Students thus worked autonomously to assess the test efficiency in terms of limit of detection (LOD, the minimum concentration of adenosine detected) by assaying a series of diluted solutions containing adenosine. The assay specificity was confirmed with control experiments carried out with an alternative nucleoside (uridine) analogue to the target (adenosine). A positive control for the effective derivatization of the gold nanoparticles with oligonucleotides was also run in parallel by hybridizing complementary DNA sequences. This control step was used to confirm the proper nanoparticle functionalization with the oligonucleotides. For this reason, this sample was also considered as a positive control for AuNPs aggregation. This practical then helped the students to become familiar with the major issues in the development of any sensor or biosensor (e.g., LOD, specificity, and positivity assessments), along with the design of appropriate controls for the qualification of the assay components.

aptamer sequence has been fully characterized and used in a variety of biosensors.10−12 Interestingly, only a few examples have been published so far employing a combined use in a detection assay of both adenosine aptamers and nanoparticles.13−15

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EDUCATIONAL PURPOSES OF THE PRACTICAL SESSION This practical session was an opportunity for the students to widen their experience and knowledge of two domains of applied biochemistry. First, students explored original properties of synthetic oligodeoxynucleotide aptamers. This experience emphasized the interest in using short DNA strands for applications other than the native genetic data storage function of genomic DNA. This noncanonical functionality of DNA, i.e., its ability to be used as a molecular probe for non-nucleotide targets, shows promise in DNA-based biosensing and other nanotechnologies.16−18 In the case of DNA aptamers, the sequence-controlled 3D folding of the oligodeoxynucleotides ensures the production of a reproducible structure exhibiting remarkable specificity and sensitivity to a molecular target of interest.6,7 This practical experienced enabled the students to grasp this concept by immobilizing DNA aptamers on the surface of gold nanoparticles (AuNPs) for biosensing purposes. A large number of emerging applications and papers describing the use of DNA or RNA aptamers for sensing applications are regularly published. This work illustrates the competitive cost of synthetic oligonucleotides, their easy chemical functionalization, and their high potential for detection of targets smaller than 1000 Da, which represents the lower limit of detection of antibody-based immune-assays. This increasing interest for the operation of nucleic acids as molecular sensing probes is counterbalanced by the highly challenging selection of efficient aptameric sequences, which represents the main bottleneck in the extended use of aptamers for biosensing. This issue partly explains why commercial products based on aptamers are still missing to date.19 Second, this practical involved surface chemistry of nanoobjects (20 nm diameter gold nanoparticles) decorated with split aptamers bound to their surface. Such a nanomaterial is easy to synthesize,20,21 cost-effective, chemically stable,22 and quite safe.23 Recently, the chemical engineering of AuNPs paved the way for an incredibly large panel of bioassays devoted to diagnostic purposes.24 Chad Mirkin’s group pioneered this field by describing simple-to-operate assays involving AuNPs, functionalized by DNA fragments forming self-assembled monolayers on the nanomaterial surface.25−27 This process is based on the high affinity of thiolated species toward gold surfaces, which eventually form compact molecular layers on gold-covered materials. This simple, straightforward, and selective route has been used in this laboratory training for the covalent and oriented assembly of thiolated oligonucleotides on AuNPs, and could easily be extended to other gold-covered materials. The antiadenosine aptamer has been chosen as a model system for this practical session due to its efficiency for adenosine capture and thorough characterization of its small molecule binding in the literature.8,10,12,14 More recently, the original antiadenosine sequence has been cut into two “split aptamers” that self-assemble in the presence of adenosine.13,15 The present practical was based on a similar strategy but was improved and adapted to match the two-day practical sessions.

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EXPERIMENTAL PROCEDURE All organic reagents and salts were purchased from SigmaAldrich (St Louis, MI, USA, see Table S1, page S2 in the Supporting Information). AuNPs (20 nm diameter) may be synthesized by the students themselves (refer to the instructors’ notes in the Supporting Information), or be purchased from several suppliers. The nanoparticles used in this practical were citrate-coated and ordered from BBI Solutions (Cardiff, UK). Although citrate-coated gold nanoparticles were quite stable in aqueous solutions, some spontaneous irreversible aggregation might be observed upon covalent modification of the nanoparticles. For this reason, before conjugation with the split aptamer oligonucleotides, the citrate ligand was preferably exchanged by a phosphine ligand according to a protocol described elsewhere.28 The phosphine ligand (bis(p-sulfonatophenyl)-phenylphosphine (BSPP)) was suspended in deionized water before citrate exchange. Oligonucleotide sequences were designed with a thiol moiety on the 5′-end. This functional group is linked through a C6 arm to the very last phosphate group of each sequence. Then, a series of 10 thymines was inserted in the 5′-end to act as a spacer between the nanoparticle surface and each aptamer domain (Table 1). The spontaneous and selective chemical functionalization of the AuNPs relies on the fast coupling (within a few minutes) of Table 1. Oligonucleotide Sequences Used for the AuNPs Functionalization Name

Sequence (5′ → 3′)a

ε (M−1 cm−1) at 260 nm

APT-ADE4 APT-ADE8 cutAPT-ADE

HS-T10-TGCGGAGGAAGGTAGAG HS-T10-TGCGGAGGAAGGTTCTC HS-T10-AGAGAACCTGGGGGAGTAT

261,700 245,000 280,000

a

HS labeling stands for the thiol moiety inserted on the 5′-end. T10 corresponds to a series of 10 thymines used as a spacer. B


Journal of Chemical Education sulfur atoms present in thiol or disulfide moieties, with Au atoms present on the AuNPs’ surface. The concentration assessments of the oligonucleotide sequences and of colloidal AuNPs’ aqueous solutions were carried out using a UV−vis spectrophotometer recording the optical density (OD) at 260 nm (the wavelength of maximum absorbance of nucleic bases) and 520 nm (the maximum plasmonic absorbance of 20 nm diameter AuNPs), respectively. For that, we used the generally agreed upon20 extinction coefficient at 520 nm for 20 nm diameter nanoparticles: ε = 8.6 × 108 M−1 cm−1. Students thus completed the formation of self-assembled-monolayers (SAMs) on the Au surfaces in a 100 μL total volume, by mixing (in the following order): 10 μL of BSPP stock solution (5 mg/100 μL of pure water), AuNPs from a stock solution (by adjusting the volume so that the final concentration in 100 μL had an OD of 10 at 520 nm), HEPES-based 5× buffer (50 mM HEPES, 25 mM MgCl2, 750 mM NaCl, 0.025% Tween 20, pH 7.4) so that the final concentration was HEPES-based 1× buffer (5 times dilution), and then 10 μL of oligonucleotide at 10 μM in pure water (1 μM final concentration for each oligonucleotide). Then, samples were kept at room temperature for 1 h to allow self-assembly of the thiolated oligonucleotides on the AuNP surfaces. During this time, students prepared a 10 mL solution of HEPES 1× buffer containing 10 μM of thiolated-PEG (poly(ethylene glycol)methyl-ether-thiol, MW 2000 Da; supplied by Sigma-Aldrich). This reagent was used to saturate any free Au-binding sites remaining on the nanoparticle surface. After the coupling between the oligonucleotides and the nanoparticles was completed, 1 mL of HEPES/thiolated-PEG solution was added to each sample and left on the bench for 15 min. Then, all samples were centrifuged in Eppendorf tubes at 10,000g for 5 min to precipitate the nanoparticles. Students had to carefully remove the supernatant with an automatic pipet, followed by the resuspension of the pellet of AuNPs in 500 μL of 1× HEPES buffer. This step was repeated twice to ensure the complete removal of any unreacted thiolated species. After discarding the last volume of supernatant, AuNPs-oligonucleotides were suspended in 1× HEPES buffer (200 μL for APTADE4 and APT-ADE8, and 400 μL for cutAPT-ADE), and sample volumes were adjusted so that the OD at 520 nm could be easily monitored (see Supporting Information). Stock solutions of adenosine and uridine (Figure 1) were prepared in HEPES 1× buffer (10 mM stock solution for each

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HAZARDS

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RESULTS AND DISCUSSION


The whole laboratory training should be done wearing personal protective equipment such as gloves, safety glasses, and a lab coat. In case of AuNP synthesis by Au(III) reduction by citrate ions, specific attention should be devoted to the handling of tetrachloroauric acid (III), which is highly corrosive. Regarding the AuNP synthesis, better results in terms of nanoparticle size monodispersity were obtained when the glassware and stir bar were first cleaned by aqua regia (CAUTION: This solution is highly corrosive and should only be used in a fume hood). It is recommended that only the instructor handle the aqua regia. After AuNP synthesis, the nanoparticles can be handled safely and the practical experience did not present any significant hazard. Indeed, others showed that gold nanoparticle doses as high as 20 mg/ kg could be administered to mice with no sign of any toxicity.23 Interestingly, gold nanoparticle colloidal solutions have also been used over the centuries for putative curative properties in humans.29 Today, gold nanoparticles are still discussed as potential therapeutic agents.30

Control of the Oligonucleotide Grafting on the Nanoparticles

Before running the adenosine bioassay, students controlled the efficient grafting of oligonucleotide sequences with the help of cutAPT-ADE and APT-ADE8 conjugates. Indeed, these sequences may spontaneously hybridize to each other at room temperature and form an 8-base-pair-long stem motif, even in the absence of adenosine, on one side of the selfassembled structure (Figure 2A). The corresponding melting temperatures were assessed using freely available online tools31 which confirmed that the melting temperatures were above room temperature, thus suggesting the stability of the expected molecular architecture, in the absence of adenosine. The control experiment was done in a 200 μL volume by mixing the cutAPT-ADE and APT-ADE8 nanoparticle derivatives at nanomolar levels (1 nM as the final concentration for each species). The total volume was adjusted by adding HEPES 1× buffer. As the self-assembly is favored by the higher sample concentration in the bottom of the vial, all samples were centrifuged at 15,000g for 5 min at room temperature. If the nanoparticles were properly functionalized with both oligonucleotides, they hybridized to each other, aggregated, and then formed a dark pellet settling at the bottom of the vial (Figures 3 and 4). If the species did not selfassemble, the gold nanoparticles were easily resuspended by gentle shaking, giving the deep red color initially visible in the vial. The self-aggregation process may also occur at room temperature, without centrifugation, within a few tens of minutes. The thermal stability of such a molecular architecture was then explored by proposing to the students to heat the sample until the double-stranded domains dissociate and then form the same red colloidal suspension as in the initial state. Each group of students chose one aggregated sample and then dipped the vial in hot tap water (around 50−60 °C). After only a few seconds, a gentle shaking of the sample enabled the total resuspension of the aggregated pellet into a red colloidal solution.

Figure 1. Ribonucleosides used for the bioassay

nucleoside) the day before the practical sessions. Regarding the specificity test, guanosine might be considered as a more relevant negative control than uridine as its molecular structure is closer to adenosine, but due to guanosine’s poor solubility in water at millimolar levels, uridine was preferred as the negative control. C




Figure 3. Absorbance measurements at 520 nm of the different samples. The first sample (Control ADE8/cut) corresponds to the double-stranded structure between nanoparticles functionalized with APT-ADE8 and cutAPT-ADE. Due to its aggregation and precipitation, there was no absorbance left in the supernatant at 520 nm. The other samples contained the APT-ADE4/cutAPT-ADE conjugates and different concentrations of nucleoside (adenine or uridine).

Detection of Adenosine and Assessment of the LOD

Figure 4. Photographic images of the gold nanoparticles functionalized with the aptamers, in the presence/absence of nucleosides. All images have been taken after sample centrifugation followed by gentle shaking by hand to control the aggregation. On the left, the sample contained the cutAPT-ADE and APT-ADE8 conjugates that selfhybridized to each other, even in the absence of adenosine. The aggregated nanoparticles were forming a dark pellet that could not be resuspended at room temperature. The sample on the left-hand side was used as a positive control of gold nanoparticle oligodeoxynucleotide functionalization. The sample in the middle contained the cutAPT-ADE and APT-ADE4 conjugates that self-assembled only in the presence of adenosine. The sample on the right contained the cutAPT-ADE and APT-ADE4 conjugates in the presence of uridine that did not induce nanoparticle aggregation. After centrifugation and gentle shaking, the red colloidal solution was obvious.

For the detection of adenosine, the assay was carried out with a cutAPT-ADE/APT-ADE4 mixture, and the final reaction volume was set at 500 μL (to be completed with HEPES 1× buffer). The final concentration of each nanoparticle derivative was 1 nM. For the assessment of the LOD, we chose to start from 1 μM up to 5 mM final concentration of adenosine (use preferably 10× adenosine stock solutions for each condition). Each group of students prepared a series of 1.5 mL Eppendorf tubes, with one tube for each adenosine concentration to be tested. After addition of cutAPT-ADE and APT-ADE4 nanoparticles, 20 μL of adenosine 10× stock solutions (10 μM, 100 μM, and 1 mM) were deposited into each tube, respectively, and the volume was adjusted to 200 μL with HEPES 1× buffer. Similar to the control experiment, the adenosine binding, and thus the nanoparticle aggregation, was sped up by sample centrifugation at 15,000g for 5 min at room temperature. After centrifugation, students recovered all samples with a dark pellet lying at the bottom of the tube in a colorless solution. Then, each vial was gently shaken before

measuring the OD520nm. If the sample contained a sufficient amount of adenosine to trigger the aggregation (meaning the adenosine concentration was higher than the LOD), nanoparticles could not be resuspended at room temperature, and students observed the persistence of the pellet in a colorless solution. If samples did not contain adenosine or contained adenosine at concentrations lower than the LOD (for instance 0.1 to 10 μM), then students could recover the red colloidal solution by shaking the sample (no vortex needed). Aggregation of AuNPs was followed in an instrument-free manner by assaying a series of adenosine solutions from 0.1 μM to 5 mM (0.1 μm, 1 μM, 10 μM, 100 μM, 1 mM, and 5 mM). Students could determine that the LOD of such a bioassay was in the range from 10 μM to 1 mM. Nevertheless, absorbance measurements at 520 nm with a UV spectrophotometer enabled a more accurate measure of the LOD (Figure 3). Indeed, with this optical analysis, students could easily confirm that the LOD value is between 10 and 100 μM of

Figure 2. Gold nanoparticles self-assembling in control and bioassays. (A) A control experiment involving AuNPs functionalized with cutAPT-ADE and APT-ADE8 was carried out to confirm the efficient self-assembly of the oligonucleotides on the surface of AuNPs. This aggregation was induced by the hybridization of an 8-base-pair sequence involving both DNA strands (shown in the red frame). (B) Bioassay describing the self-assembling of cutAPT-ADE and APTADE4 in the presence of adenosine, but not with uridine.

D




(Labex Arcane, ANR-11-LABX-0003-01). Y.R. also thanks Larry O’Connell for its help in the manuscript preparation.

adenosine. If the adenosine concentration was lower than the LOD, then the nanoparticles were easily resuspended and gave the deep-red colloidal solution.

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Assessment of the Specificity of the Adenosine Bioassay versus Uridine

(1) European School On Nanosciences & Nanotechnologies. https://www.esonn.fr (accessed March 6, 2019). (2) Ralevic, V.; Burnstock, G. Receptors for purines and pyrimidines. Pharmacol Rev. 1998, 50 (3), 413−492. (3) Holgate, S. T. Airway inflammation and remodeling in asthma: current concepts. Mol. Biotechnol. 2002, 22 (2), 179−189. (4) Yaar, R.; Jones, M. R.; Chen, J. F.; Ravid, K. Animal models for the study of adenosine receptor function. J. Cell. Physiol. 2005, 202 (1), 9−20. (5) Sollevi, A. Cardiovascular effects of adenosine in man; possible clinical implications. Prog. Neurobiol. 1986, 27 (4), 319−349. (6) Ellington, A. D.; Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346 (6287), 818− 822. (7) Tuerk, C.; Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990, 249 (4968), 505−510. (8) Huizenga, D. E.; Szostak, J. W. A DNA aptamer that binds adenosine and ATP. Biochemistry 1995, 34 (2), 656−665. (9) Perez-Ruiz, T.; Martinez-Lozano, C.; Tomas, V.; Martin, J. Determination of ATP via the photochemical generation of hydrogen peroxide using flow injection luminol chemiluminescence detection. Anal. Bioanal. Chem. 2003, 377 (1), 189−194. (10) Chen, S. J.; Huang, Y. F.; Huang, C. C.; Lee, K. H.; Lin, Z. H.; Chang, H. T. Colorimetric determination of urinary adenosine using aptamer-modified gold nanoparticles. Biosens. Bioelectron. 2008, 23 (11), 1749−1753. (11) Huang, D. W.; Niu, C. G.; Qin, P. Z.; Ruan, M.; Zeng, G. M. Time-resolved fluorescence aptamer-based sandwich assay for thrombin detection. Talanta 2010, 83 (1), 185−189. (12) Kim, J.; Kim, I. Y.; Choi, M. S.; Wu, Q. Label-free electrochemical detection of adenosine based on electron transfer from guanine bases in an adenosine-sensitive aptamer. Chem. Commun. (Cambridge, U. K.) 2009, No. 31, 4747−4749. (13) Li, F.; Zhang, J.; Cao, X.; Wang, L.; Li, D.; Song, S.; Ye, B.; Fan, C. Adenosine detection by using gold nanoparticles and designed aptamer sequences. Analyst 2009, 134 (7), 1355−1360. (14) Melaine, F.; Coilhac, C.; Roupioz, Y.; Buhot, A. A nanoparticlebased thermo-dynamic aptasensor for small molecule detection. Nanoscale 2016, 8 (38), 16947−16954. (15) Melaine, F.; Roupioz, Y.; Buhot, A. Gold Nanoparticles Surface Plasmon Resonance Enhanced Signal for the Detection of Small Molecules on Split-Aptamer Microarrays (Small Molecules Detection from Split-Aptamers). Microarrays 2015, 4, 41−52. (16) Wang, P.; Meyer, T. A.; Pan, V.; Dutta, P. K.; Ke, Y. The Beauty and Utility of DNA Origami. Chem. 2017, 2 (3), 359−382. (17) Hong, F.; Zhang, F.; Liu, Y.; Yan, H. DNA Origami: Scaffolds for Creating Higher Order Structures. Chem. Rev. 2017, 117 (20), 12584−12640. (18) Veneziano, R.; Ratanalert, S.; Zhang, K.; Zhang, F.; Yan, H.; Chiu, W.; Bathe, M. Designer nanoscale DNA assemblies programmed from the top down. Science 2016, 352 (6293), 1534. (19) Baird, G. S. Where are all the aptamers? Am. J. Clin. Pathol. 2010, 134 (4), 529−531. (20) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Preparation and Characterization of Au Colloid Monolayers. Anal. Chem. 1995, 67 (4), 735−743. (21) Sharma, R. K.; Gulati, S.; Mehta, S. Preparation of Gold Nanoparticles Using Tea: A Green Chemistry Experiment. J. Chem. Educ. 2012, 89 (10), 1316−1318. (22) Lee, C. F.; You, P. Y.; Lin, Y. C.; Hsu, T. L.; Cheng, P. Y.; Wu, Y. X.; Tseng, C. S.; Chen, S. W.; Chang, H. P.; Lin, Y. W. Exploring the Stability of Gold Nanoparticles by Experimenting with Adsorption Interactions of Nanomaterials in an Undergraduate Lab. J. Chem. Educ. 2015, 92 (6), 1066−1070.

Assessment of specificity to a target is a key point in the development of sensors or biosensors. Control experiments must be carried with alternative biomolecules, similar to the target, and processed in similar conditions to appraise this parameter. In the present assay, uridine, which is another ribonucleoside (Figure 1), was chosen as a control. The assay with cutAPT-ADE/APT-ADE4-functionalized nanoparticles was then repeated in the presence of uridine, instead of adenosine, with 10 mM uridine solutions (note that millimolar concentrations of uridine, from 1 to 5 mM, were used without an effect on the aggregation as seen in Figures 3 and 4). Once again, all samples were centrifuged (15,000g for 5 min at room temperature) to favor the nanoparticle aggregation.

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CONCLUSIONS In conclusion, a laboratory protocol was described which was split into two 4 h sessions over two consecutive days. This protocol appealed to a series of chemical competencies, ranging from metallic nanoparticle handling, surface functionalization and characterization, to the use of short synthetic DNA strands for biosensing purposes. The students could set their own bioassay and successfully managed to assess its performance in terms of sensitivity and specificity for a predetermined metabolic biomarker. This laboratory training required safe and affordable reagents as well as routine equipment (centrifuge and UV−vis spectrophotometer). Interestingly, further aspects of nanoparticles and nucleic acids reactivity were also explored, with such material in hand, such as DNA self-assembling reversibility. Whatever the scientific background of the students (be they physicists, chemists, or biotechnologists), they all managed to prepare their own set of DNA-functionalized gold nanoparticles, and they gained confidence and familiarity in the use of colloidal nanomaterials for bioinspired applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.9b00044. Information on material and reagents, details on experimental protocols, and UV−vis quantification of gold nanoparticles and oligodeoxynucleotides (PDF, DOCX)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yoann Roupioz: 0000-0002-1790-8141 Notes

The author declares no competing financial interest.

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ACKNOWLEDGMENTS This work has been partly funded by the GDRi NanoAndes and also by the French Laboratory of Excellence ANR program E




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Functionalization of Gold Nanoparticles for a Color-Based Detection of

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