Portable Detection of Melamine in Milk Using a Personal Glucose

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Portable Detection of Melamine in Milk Using a Personal Glucose Meter Based on an In Vitro Selected Structure-Switching Aptamer Chunmei Gu, Tian Lan, Han-Chang Shi, and Yi Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01085 • Publication Date (Web): 06 Jul 2015 Downloaded from http://pubs.acs.org on July 11, 2015

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Analytical Chemistry

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Portable Detection of Melamine in Milk Using a

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Personal Glucose Meter Based on an In Vitro

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Selected Structure-Switching Aptamer

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†‡ ‡ † ‡, Chunmei Gu ¶, Tian Lan ¶, Hanchang Shi *, Yi Lu §,*

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†

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Beijing 100084, P. R. China

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‡

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Urbana-Champaign, Urbana, Illinois 61801, USA

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Corresponding author: [email protected], [email protected].

State Key Joint Laboratory of ESPC, School of Environment, Tsinghua University,

Department of Biochemistry, §Department of Chemistry, University of Illinois at

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ACS Paragon Plus Environment

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ABSTRACT:

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Melamine detection in milk and other foods has attracted much attention since the

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discovery that melamine-adulterated food causes severe kidney damage. Although many

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methods have been developed to detect melamine, few methods can provide quantitative

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results using an affordable and portable device that is suitable for home use or field

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application. To achieve this goal, we herein report the first in vitro selection of a

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melamine responsive aptamer using a structure-switching method. A personal glucose

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meter based melamine sensor was designed and subsequently tested using the newly

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isolated aptamer. Conversion of melamine concentration to glucose amount was achieved

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by including an invertase-conjugated DNA that is complementary to part of the aptamer.

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Melamine binding triggers the release of the invertase-DNA conjugate, which hydrolyzes

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sucrose into glucose. The glucose produced is then measured directly using an off-the-

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shelf personal glucose meter. The described sensor shows high selectivity for melamine

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against several closely related melamine analogues, such as cyanuric acid, ammeline and

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ammelide, and has low detection limits of 0.33 µM (or 41.1 ppb) in buffer and 0.53 µM

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(or 67.5 ppb) in 80% whole milk without any pretreatment. The detection limits meet the

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threshold of 2.5 ppm for non-infant-formula products and 1 ppm for melamine in infant

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milk products as defined by the US Federal Food and Drug Administration (FDA). In

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addition to the PGM sensor demonstrated here, the same aptamer can be converted into

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other types of sensors with different signal outputs, allowing portable detection of

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melamine under a variety of conditions.

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Key Words: SELEX; Structure-Switching aptamer; melamine; portable detection;

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personal glucose meter (PGM); milk

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Introduction

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Melamine adulteration in milk products has caused thousands of renal failure cases and

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several infant deaths in China.1,2 As a result, different government agencies have imposed

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strict regulations on limits of detectable melamine in food and milk products. In the U.S.,

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the Federal Food and Drug Administration (FDA) have established a threshold of 2.5

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ppm for melamine for non-infant-formula products and a 1 ppm threshold in infant milk

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products.3 Currently, detection of melamine in various food sources can be carried out

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using standard analytical instruments, such as mass spectroscopy2,4-7 and high

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performance liquid chromatography.6,8,9 Although these techniques can achieve detection

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limits down to ppb, they require the use of expensive instruments, careful calibration and

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a long waiting time to obtain results, making them unsuitable for on-site and real-time

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detection. To overcome these limitations tests using immunoassays,10,11 nanoparticle

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based methods12-16 and microfluidic electrophoresis device (MED)-UV detection17 have

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shown promise. However, to make these methods field deployable, one still needs an

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affordable and portable meter, because melamine detection requires quantitative results.18

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While a number of portable instruments have been developed, most of them are costly to

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design and market and are therefore not affordable for common use.18-21 Many of them

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are also not pocketable or easy to operate, hence difficult for wide adoption. Recently we

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have adopted the widely available personal glucose meter (PGM) as a general meter for

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detecting of wide range of non-glucose targets.18 By bypassing costly meter design by


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using the small, inexpensive, and accessible PGM, we developed a melamine sensor that

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is not only portable and cost effective, but also easy to use.

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The key to adapting the PGM to detect other targets is to incorporate an intermediate step

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linking the binding of either antibody or aptamer to its targets with the production of

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glucose using invertase, which is capable of hydrolyzing sucrose to glucose.18,22 In doing

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so, we can establish a direct relationship between the presence and concentration of the

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target and the presence and concentration of glucose. While an antibody for melamine is

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available, its high cost and low stability curtail its use in portable and affordable

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melamine sensors.23

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Isolated via a technique called Systematic Evolution of Ligands by Exponential

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enrichment (SELEX),25,26 aptamers are DNA or RNA sequences that can bind tightly and

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selectively to desired targets, such as proteins, small molecules, metal ions, even whole

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cells.25-31 Compared to antibodies, aptamers have distinct advantages including fast,

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reproducible synthesis and greater stability than antibodies.24,32,33 Moreover, for sensing

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applications, DNA aptamers are superior to antibodies at transforming the binding event

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into detectable signal, because aptamers can significantly change their structure upon

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binding to a target in a predictable manner and are much easier to conjugate to signaling

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agents, such as chromophores, fluorophores or electrochemical agents.32-35 However, for

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biosensing applications, modifications are generally required to convert an aptamer to a

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sensor. To achieve a convenient conversion from aptamer to sensor, Li and co-workers

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have employed a structure-switching selection method that utilizes the conformational

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change (structure-switching) of the aptamer from target-free to target-bound state as one

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of the selection conditions.36


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Herein, we report the use of the structure-switching selection method to obtain a

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melamine aptamer for biosensing application. After successful isolation of the melamine-

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responsive aptamer, a portable sensor was developed using PGM for portable

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quantification18,22,37-39 of melamine in milk.

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EXPERIMENTAL SECTION

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Materials and Apparatus. All DNA sequences were ordered from Integrated DNA

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Technologies (Coralville, IA). General chemicals and buffers were purchased from

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Sigma (St. Louis, MO). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP),

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sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), and

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ScintiVerse™ BD Cocktail were from Fisher Scientific (Pittsburgh, PA). [γ-32P]-ATP

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and [α-32P]-ATP were purchased from PerkinElmer (Shelton, CT). 40% Acrylamide/Bis

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Solution (29:1) was from Bio-Rad (Hercules, CA). All solutions were prepared using

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Milli-Q water with electrical resistance over 18 MΩ·cm. DyNamo SYBR Green qPCR

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Kits were purchased from New England Biolabs Inc. (Ipswich, MA). Avidin resin was

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purchased from PIERCE Inc. (Rockford, IL). T4 polynucleotide kinase, Platinum Taq

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polymerase, TOPO® TA Cloning Kit, and Dynabeads® MyOne™ Streptavidin C1

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magnetic beads were purchased from Invitrogen (Carlsbad, CA). Amicon centrifugal

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filters were purchased from Millipore Inc. (Billerica, MA). The LS6500 multi-purpose

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scintillation counter was from Beckman Coulter Inc. (Brea, CA). The STORM 840

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scanner and phosphoimager cassette for gel imaging were from Molecular Dynamic

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(Sunnyvale, CA). The Breeze2® glucose meter was from Bayer HealthCare LLC

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(Tarrytown, NY).


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Procedures for Each Selection Round. The DNA sequences used are shown in Table

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S1. Briefly, 1 µL of the starting pool, before each round of selection, was saved for liquid

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scintillation counting. For the first round of selection, 3 nmol of random DNA was

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labeled with [γ-32P]-ATP using kinase reaction. The radiolabeled DNA pool was

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annealed to BDNA (biotinylated DNA), P1 and P2 for 1 hr before it was added to 300 µL

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of avidin resin, which has been washed 10 time with 0.3 mL 1xSB (Selection Buffer, 100

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mM NaCl, 5 mM KCl, 5 mM MgCl2, 20 mM Ca(NO3)2, 100 mM NaHEPES, pH 7.0).

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The DNA was allowed to bind to the avidin column for 1 hr. After the incubation, the

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column was washed with 5 mL 1xSB and 10 fractions of 500 µL, wash fractions were

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collected. After washing, the DNA was incubated on the column for 1 hr to estimate the

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background. After the incubation without target, the column was washed with 1 mL

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1xSB and 2 fractions of 500 µL buffer were collected and served as the background for

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calculating the switching activity. A total of 300 µL avidin resin was left in the column

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before the addition of melamine. A small volume of 25 mM melamine was added to the

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DNA to achieve the target elution concentration for that round. The DNA library was

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incubated with melamine for 1 hr (the same length of time as the background washes),

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then eluted with 1xSB with 100 µM melamine (target elution fractions). Five target

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elution fractions were collected and the first two fractions were counted by liquid

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scintillation.

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as (

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was amplified by PCR using one primer with a 3′ RNA. The PCR product was

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hydrolyzed with 6 M NaOH and neutralized before separating on a 10% denaturing

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PAGE along with appropriate oligonucleotide markers. The band with the appropriate

The

switching

activity

(S.A.)

was

calculated

. Ten microliter of the first target elution fraction

! " )


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length was cut out and the DNA was extracted then desalted by ethanol precipitation. The

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purified DNA pool was used for next round of selection.

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Cloning and Sequence Alignment. 10 µL of the gel purified DNA pool was amplified

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by PCR and purified with the QIAquick PCR purification kit (Qiagen). Standard cloning

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procedure for TOPO® TA cloning (Invitrogen) was followed to obtain plasmids from 96

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individual colonies for sequencing. Sequence alignment was performed with VectorNTI

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(Invitrogen).

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Aptamer Identification and Re-selection. Selected sequences identified from cloning

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were tested for their switching activity in a manner similar to the method used during the

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selection rounds. Briefly, each clone to be tested was obtained by PCR amplification with

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[α32-P] ATP and the RNA containing primer. After hydrolyzing the RNA base and

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neutralization, the desired aptamer strand was separated on a 10% denaturing PAGE then

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gel extracted and desalted by ethanol precipitation. Approximately 30 pmol of each clone

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was then tested for its S.A. similar to the selection procedure. Briefly, 30 pmol of each

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clone was allowed to bind with 150 pmol BDNA, P1 and P2 for 1 hr. After binding, the

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DNA was added to avidin resin and allowed 1 hr for binding. The avidin resin was then

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washed with 1xSB. Background and target elution fractions were then collected to

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calculate the switching activity.

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The re-selection pool was designed by re-randomizing Class V with –P2 truncation

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(sequence shown in Table S2). Each of the nucleotides positioned in the N20 and N30

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regions had a 20% chance of being substitute with the other three nucleotides (20%

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degeneracy) when the re-selection pool was ordered from Integrated DNA Technologies.


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Primers and other DNA sequences used in the re-selection are shown in Table S2.

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Experimental procedure for the re-selection was identical to the original selection. The

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new selection buffer (1xNSB) contained 50 mM NaCl, 0.5 mM MgCl2, 0.5 mM

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Ca(NO3)2, 50 mM NaHEPES at pH 7.0.

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Isothermal Titration Calorimetry (ITC). ITC was performed using a MicroCal iTC200

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(Malvern, Sweden) instrument. Data was fit to a one site binding model using Origin 7.0

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software. Samples were centrifuged to remove particles before loading into the ITC cell

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and syringe. All experiments were corrected for the heat of dilution of the titrant. Unless

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otherwise specified, the targets and aptamer solutions were prepared in 1xNSB. The

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binding experiments were performed with aptamer solutions ranging from 10 to 20 µM

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using ligand concentrations of 0.125 − 1 mM at 25℃. All titrations were performed with

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the aptamer in the cell and with the ligand, as the titrant, in the syringe. The standard

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binding experiments consisted of 16 successive 2.4 µL injections every 100s. The first

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injection was 0.2 µL. Dissociation constants were calculated from the association

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constants measured by ITC.

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DNA-Invertase Conjugation. The conjugation procedure was similar to our previous

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work with some minor modifications.18,37,39 Briefly, 30 µL of 1 mM thiol-DNA, 2 µL of 1

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M sodium phosphate buffer at pH 5.5, and 2 µL of 30 mM TCEP were mixed and kept at

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room temperature for 1 hour. After that, the excess TCEP was removed by Amicon-3K

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using Buffer A (0.1 M NaCl, 0.1 M sodium phosphate buffer, pH 7.3, 0.05% Tween-20)

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without Tween-20 for 8 times. Meanwhile, 1 mg of sulfo-SMCC was mixed with 400 µL

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of 20 mg/mL invertase in Buffer A without Tween-20. After vortexing, the solution was

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placed on a shaker for 1 hour at room temperature. Then the mixture was centrifuged to


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remove excess insoluble sulfo-SMCC. The supernatant was washed 8 times by Amicon-

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100K using Buffer A without Tween-20. The above solutions of TCEP-activated thiol-

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DNA and sulfo-SMCC-activated invertase were mixed and kept at room temperature for

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48 hours. To remove unreacted thiol-DNA, the solution was purified by Amicon-100K

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using Buffer A without Tween-20 and gradually changing to melamine selection buffer

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over eight wash cycles.

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Procedures for Using the PGM to Detect Melamine. Firstly, 300 pmol Rd29C33, 300

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pmol BDNA, 300 pmol invertase-DNA were mixed together in 100 µL 1xNSB at room

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temperature for 30 minutes. The solution was then mixed with 240 µL streptavidin coated

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magnetic beads (MBs), which had been buffer-exchanged to 1xNSB at room temperature,

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for 30 minutes. After binding to MBs, they were washed with 1xNSB five times and then

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separated into portions of 20 µL MBs per each Eppendorf tube. Then, 40 µL of solution,

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containing varying melamine concentrations in 1xNSB or milk, was added to the 20 µL

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MBs Eppendorf tubes and vortexed for 10 minutes at room temperature. After mixing

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and being left to sit on a magnetic strand, 15 µL of the supernatant was added to 5 µL of

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2M sucrose and allowed it to react at room temperature for 20 minutes. The glucose

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content was measured using the PGM (Breeze2®, Bayer).

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RESULTS AND DISCUSSION

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SELEX procedure and PGM sensor design.

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The selection design for aptamers for melamine is based on the structure-switching

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SELEX technique reported by Li and coworkers (Scheme 1a).36 The conversion from

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isolated structure-switching aptamers to a PGM based melamine sensor is achieved by


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addition of an invertase-conjugated DNA that hybridizes with the aptamer (Scheme 1b).

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Both the SELEX procedure and the PGM sensor rely on the structure-switching of the

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aptamer upon binding to melamine. This binding event disrupts DNA hybridization

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between the aptamer to an anchoring DNA strand and allows its subsequent release from

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the solid support.

201 202

Scheme 1. Schematic of the (a) SELEX procedure and (b) PGM sensor design. The

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SELEX procedure was adapted from previously published method. To convert the

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isolated aptamer to a PGM-based sensor, invertase-conjugated DNA was added to the

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DNA sequences used in SELEX. Upon binding to melamine, the structural changed of

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the isolated aptamers resulted in release of the invertase-conjugated DNA, which can

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generate a signal that is proportional to the amount of melamine present and can be

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measured by PGM.

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Selection of Aptamers for Melamine Using Structure-Switching Method. The random

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DNA library was designed based on a structure-switching model reported by Li and

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coworkers.36 A total of 17 rounds of selection were performed in 1xSB after a 1 hr


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incubation and then elution with melamine. The initial melamine concentration for the

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incubation and elution was kept at 0.5 mM (round 1–10), then reduced gradually to 20

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µM towards the end of the selection. The selection progress was monitored by calculating

215

the switching activity (S.A.) for each round. Figure 1 shows the progression of S.A.

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during the selection: at round 8, S.A. began to rise and peaked at round 10. More

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stringent conditions were then introduced at round 11 by reducing the eluting melamine

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concentration to 50 µM. The S.A. at round 11 decreased to 3.34 but recovered to 4.9 in

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round 12. To further increase the selection stringency, melamine concentration was

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further reduced to 20 µM from round 13 to 17, an initial decrease of S.A. was observed at

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round 13 but recovered by round 17. After round 12, three rounds of parallel selections

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were also conducted, where 1% milk was introduced to the selection buffer. The activity

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of the pool was maintained with the addition of milk, indicating the isolated aptamers had

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the potential to perform in milk.

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Figure 1. Selection profile for SELEX of aptamers for melamine using the structure-

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switching method. Melamine concentration started at 500 µM from round 1 to round 10

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and was reduced to 50 µM at round 11 to round 12. From round 13, melamine


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concentration was reduced to 20 µM and was branched into two parallel selections (no

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milk and 1% milk). Incubation time was kept at 1 hour throughout the selection process.

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As no apparent switching activity increase was observed after round 13, the DNA pool

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for round 13 was cloned and sequenced. After sequence alignment, we found seven

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classes of clones, in addition to two quadruplicates (called Quad I and Quad II) and other

234

orphan sequences (Figure S1). Initially, both the full length and truncated versions of

235

Class I – VII were tested with 10 min incubation time with 20 µM melamine. Switching

236

activity was observed only for the full length and truncated Class V, with full length

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version showing higher activity (Table S3). In search for more candidate aptamers, the

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Quad I and Quad II aptamers were tested, and the Quad II aptamers showed similar

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activity to the full length aptamer of Class V. Therefore, we investigated the recognition

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of Class V and Quad II aptamers towards melamine by comparing their S.A. in the

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presence of two structural analogues of melamine, 6-methyl-1,3,5-triazine-2,4-diamine

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and 2,4,6-triaminopyrimidine. One of the amines in each of the analogous was replaced

243

by a carbon, resulting in the loss of a hydrogen bond at that location. Since melamine

244

only has two types of amines that are capable of forming hydrogen bonds, any loss of

245

activity associated with these amines can be used to deduce structural-functional

246

relationship. As shown in Figure 2, the Class V aptamer displayed a slightly higher

247

selectivity against the two melamine analogues. Based on the predicted secondary

248

structure of Class V aptamer, we determined the S.A. for various truncated and mutated

249

versions of the Class V aptamer (sequences shown in Table S4). As shown in Figure S2,

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most of truncations and mutations abolished S.A., except for the –P2 truncation (where

251

the P2 binding site was removed). While these results are encouraging, the Class V


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aptamer exhibited a low activity at the melamine concentration (20 µM) need for sensing

253

application. In addition, the Class V aptamer required a high concentration of divalent

254

metal ions for activity (see Figure S2). To overcome these limitations, we carried out re-

255

selection based on the Class V aptamer.

256 257

Figure 2. Selectivity of Class V and Quad II to melamine against two melamine

258

analogues, 2,4,6-triaminopyrimidine and 6-methyl-1,3,5-triazine-2,4-diamine. Switching

259

activity was measured after a 10 min incubation with 20 µM melamine.

260

Re-selection of the Class V aptamers using a degenerate pool. Based on the sequence

261

of the –P2 truncation of the Class V aptamer, a DNA library for re-selection was

262

designed by introducing 20% sequence degeneracy into the N20 and N30 region. Since one

263

of the primer binding sites was removed, part of the sequence belonging to the N30 region

264

was used as the new primer-binding site in the re-selection. The DNA sequences used for

265

the re-selection are listed in Table S2.

266

To reduce the observed dependence on divalent metal ions for the switching activity, the

267

concentrations of Mg2+ and Ca2+ were reduced from 5 mM and 20 mM, respectively, in


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the initial selection to 0.5 mM for both in the new re-selection buffer (1xNSB).

269

Furthermore, to improve the binding affinity of the possible aptamers for melamine, a

270

relatively lower elution concentration (100 µM) was used and gradually reduced to 60

271

µM. In addition, the target elution time started at 1 hour and gradually shortened to 15

272

minutes. The selection progress was monitored similarly to the original selection. After

273

29 rounds of re-selection, the S.A. reached 4.25 in the presence of 60 µM melamine after

274

15 min melamine incubation. The pool was cloned and sequenced. From the 11 clones

275

obtained from sequencing, only one class was revealed from sequence alignment (Figure

276

S3). The clone 33 of round 29 (called R29C33 hereafter) from this class was assessed for

277

sensor development. The S.A. of R29C33 at different concentrations of melamine was

278

measured and compared to Class V (–P2 truncation) aptamer in Figure 3a (in their

279

respective selection buffer). The data indicated that the R29C33 has a much better

280

activity when compared to its parent aptamer in terms of maximum signal and sensitivity.

281

Similarly, the selectivity of R29C33 was assessed with melamine analogues. As shown in

282

Figure 3b, in additional to better sensitivity than its parent sequence, R29C33

283

demonstrated a higher level of selectivity against the two structural analogues.

284

A more detailed characterization was carried out to measure the binding affinity of

285

R29C33 with melamine and various structural analogous using isothermal titration

286

calorimetry (ITC). The dissociation constant (Kd) measured by ITC for melamine was

287

0.51 ± 0.03 µM, which is significantly tighter binding than for other structural analogues

288

(Table S5 and Figure S4). A randomly chosen poly(T) sequence did not show observable

289

binding to melamine by ITC. Three hydrolysis products, cyanuric acid, ammeline and

290

ammelide, of melamine were also included in this assessment. Aptamer R29C33 has


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demonstrated roughly 1,000 or better binding to melamine than these hydrolysis products

292

that have been shown to co-exist with melamine.40 Removal of the hydrogen bonds (e.g.,

293

6-mtehyl-1,3,5-trizaine-2,4-diamine and 2,4,6-triaminopyridine) from the two different

294

type of amines significantly reduced the aptamer binding affinity, which was similar to

295

the parent sequence, Class V. These results suggest that the R29C33 aptamer is suitable

296

for biosensing applications.

297 298

Figure 3. Switching activity of Rd29C33. (a), Concentration dependent activity of

299

Rd29C33 with melamine compared to the Class V –P2 truncation, and (b), binding of

300

R29C33 to melamine at various concentrations and 6-methyl-1,3,5-triazine-2,4-diamine,

301

and 2,4,6-triaminopyrimidine.


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Portable Sensor Development Based on Rd29C33 Aptamer. Scheme 1b illustrates the

303

design of a portable sensor for melamine using the isolated Rd29C33 aptamer and a PGM.

304

To avoid decreasing the flexibility of the aptamer conjugated directly to the magnetic

305

beads (MBs), which could result in the loss of structure-switching activity, an invertase-

306

conjugated DNA was designed to hybridize to portions of R29C33 (Scheme 1b and

307

Figure 4a) to complete the sensor design. The sensor composed of R29C33 hybridized to

308

BDNA, invertase-conjugated DNA. The DNA complex was immobilized on streptavidin

309

coated magnetic beads via the biotin on the BDNA. The binding of melamine to its

310

aptamer causes a structural change of melamine aptamer, thus releasing of invertase-

311

DNA from MBs. The released invertase-DNA can then convert sucrose to glucose and

312

the final product, glucose, can be detected by PGM.

313

Because the precise location of the active site for the structure-switching activity of

314

Rd29C33

315

complementary sequences of invertase_DNA (Table S6) were screened to obtain the

316

highest structure-switching activity in order to enable sensitive detection of melamine

317

(Figure 4a). Invertase_10 nt, Invertase_12 nt, Invertase_14 nt and Invertase_16 nt (Table

318

S6), which has a complementary length of 10, 12, 14 and 16 nucleotides, respectively,

319

were tested. As shown in Figure 4b, the S.A. of Rd29C33 in presence of invertase_10nt

320

can reach 3.25 in the presence of 10 µM melamine with 5 minutes incubation, much

321

higher than other complementary lengths investigated. Interestingly, while the

322

complementary length played a key role in the S.A., the site of complementary did not

323

(Figure 4c).

was

unknown,

different

lengths

of

invertase_DNA

and

different


16

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324 325

Figure 4. Initial optimization of the sensor design. (a) Sequences used for the

326

optimization. Structure-switching activity of Rd29C33 at (b) different lengths of

327

complementary

328

invertase_DNA.

329

After optimizing the sensor constructs as shown above, we chose to incorporate

330

invertase_10nt into the PGM-based portable melamine sensor. As shown in Figure 5a, the

331

glucose signal increased with increasing concentration of melamine in buffer. A detection

332

limit of 0.33 µM melamine in buffer, 41.1 ppb, was calculated according to the definition

333

of 3σ/slope (Figure 5a). This portable sensor also showed selectivity over three melamine

334

analogues which can be found with melamine: cyanuric acid, ammeline, and ammelide.

335

As shown in Figure 5b, no detectible signal can be observed for 1 mM cyanuric acid,

336

ammeline or ammelide (column 4, 5, 6 in Figure 5b) under the same conditions used for

337

melamine (100 µM, 10 µM and 1 µM respectively for column 1, 2 and 3 in Figure 5b).

invertase_DNA,

(c)

different

shifting

site

of

complementary


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338

The invertase reaction time was increased to 2 hrs (compared to 20 min for melamine) for

339

cyanuric acid, ammeline and ammelide to allow further increase of glucose concentration.

340

Low glucose meter signals (column 7, 8 and 9 in Figure 5b) comparable to that generated

341

by 1 µM melamine can be observed. The aptamer based PGM sensor has demonstrate

342

excellent selectivity over relevant structural analogous.

343 344

Figure 5. Performance of melamine detection in buffer using a PGM. (a), Concentration

345

dependence of the PGM signal on the increasing concentration of melamine, inset is the

346

sensor performance at low melamine concentration. (b), Selectivity of the developed

347

PGM based melamine sensor against the three melamine analogues, cyanuric acid,


18

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348

ammeline and ammelide at 1 mM. Melamine concentrations tested were 100 µM, 10 µM

349

and 1 µM as shown in column 1, 2 and 3 respectively.

350

Furthermore, the applicability of this portable sensor to detect melamine in milk was

351

tested and the results are shown in Figure 6. The glucose signal increased with increasing

352

concentrations of melamine in 80% commercial whole milk without any pretreatment.

353

Based on these results, a detection limit of 0.53 µM melamine, which is 67.5 ppb, was

354

calculated according to the definition of 3σ/slope (Figure 6). Therefore, this PGM based

355

portable melamine sensor can meet the threshold of 2.5 ppm for non-infant-formula

356

products and 1 ppm for melamine in infant milk products defined by the US FDA.

357 358

Figure 6. Performance of melamine detection in 80% milk without pretreatment using a

359

PGM. Inset: sensor performance between 0 and 30 µM melamine.

360

CONCLUSIONS

361

In summary, by using the structure-switching SELEX method, a melamine responsive

362

aptamer was selected after 17 rounds of selection and 29 rounds of reselection. The

363

resulting aptamer (R29C33) showed high affinity for melamine and good selectivity


19


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Page 20 of 23

364

against closely related melamine analogues, such as cyanuric acid, ammeline and

365

ammelide, which are hydrolysis products of melamine. After truncation and optimization,

366

the Rd29C33 aptamer was converted to a portable sensor using a commercially available

367

PGM. The portable sensor exhibits concentration dependent PGM signal with detection

368

limits of 0.33 µM (or 41.1 ppb) in buffer and 0.53 µM (or 67.5 ppb) in 80% commercial

369

whole milk without pretreatment, which meet the threshold of 2.5 ppm for non-infant-

370

formula products and 1 ppm for melamine in infant milk products defined by the US

371

FDA. In addition, this structure-switching aptamer based portable sensor showed

372

excellent selectivity against melamine hydrolysis products. The R29C33 aptamer is the

373

first in vitro selected aptamer for melamine, and it can be used in other types of sensors,

374

such as fluorescent, colorimetric, and SPR sensors to for melamine detection.

375

ASSOCIATED CONTENT

376

Supporting Information

377

The supporting information contains the sequences used in in vitro selection of the

378

aptamers, truncation of the aptamers, and additional results of the PGM-based sensor.

379

These materials are available free of charge via the Internet at http://pubs.acs.org.

380

AUTHOR INFORMATION

381

Corresponding Author

382

*E-mail: [email protected], [email protected].

383

Author Contributions


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384

¶These authors contributed equally.

385

Notes

386

The authors declare no competing financial interest.

387

ACKNOWLEDGMENTS

388

We wish to thank Ms. Claire McGhee for careful proofreading of the manuscript. This

389

work has been supported by the US National Science Foundation (CTS-0120978) and

390

Major Scientific Equipment Development Project of China (2012YQ030111).

391

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Portable Detection of Melamine in Milk Using a Personal Glucose

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