Whole Blood Electrochemiluminescent Detection of Dopamine

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Article

Whole Blood Electrochemiluminescent Detection of Dopamine Alasdair J Stewart, Jodie Hendry, and Lynn Dennany Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 04 Nov 2015 Downloaded from http://pubs.acs.org on November 4, 2015

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Analytical Chemistry

Whole Blood Electrochemiluminescent Detection of Dopamine. Alasdair J. Stewart, Jodie Hendry, Lynn Dennany* WestCHEM, Department of Pure & Applied Chemistry, University of Strathclyde, Technology & Innovation Centre, 99 George Street, Glasgow, G1 1RD, UK. * [email protected], Tel: +44 (0)141 548 4322; Fax: +44 (0)141 548 2532

KEYWORDS Electrochemiluminescence, Biosensor, Dopamine, Quantum Dots, Whole Blood Detection ABSTRACT: Direct detection of medically relevant biomarkers in whole blood without the need for pretreatment or extraction is a great challenge for biomedical analysis and diagnosis. Electrochemical techniques, such as electrochemiluminescence (ECL), are promising tools for this area of analysis. ECL offers high sensitivities together with the ability to obtain time and spacial control over the process. This work exploits these features together with the low background signals obtained from ECL detection to clearly identify and quantify dopamine in whole blood with relative standard deviations lower than 5% (n = 5). This near infrared quantum dot based ECL sensor displayed a linear response over the range 3.7 ≤ [dopamine] ≤ 450 µM allowing the rapid detection of dopamine and providing a platform for future development. Significantly, the near infrared quantum dots exhibited excellent penetrability through biological samples such as whole blood, and shown the ECL detection of dopamine in whole blood for the first time. This will likely be at the forefront of development in biosensing and imaging fields in the foreseeable future.

INTRODUCTION The direct detection of biomarkers in complex biological matrices is a key health plan strategy for diagnosis of medical conditions and the subsequent monitoring of patient progress. Sensing within whole blood for point of care (POC) devices is therefore an area of significant interest in recent years. Electrochemiluminescence (ECL) has been used extensively as a detection method in bio-sensing because of its advantages over other detection techniques. Excellent sensitivity is achieved as no light source is required, resulting in minimal background light intensity1, whilst scattered light and interferences from emission by impurities or other analytes is effectively eliminated.2 Combined with the specificity of the ECL reaction, these attributes produce a technique that is ideally suited for detecting low concentration target analytes in complex matrices, such as whole blood, with a good signal to noise ratio.3-6 The potential significance of incorporating near infra-red emitting quantum dots (NIR QDs) into biosensing systems is the improved penetrative ability of light at this wavelength through whole blood and minimization of tissue autofluorescence,7,8 which may address the issues associated with the challenges of detection within whole blood. The absorption spectrum of whole blood predominately follows that of haemoglobin, which can exist in its oxygenated or de-oxygenated form.9 As the majority of clinical blood samples are venous in origin, the absorption spectrum of de-oxygenated heamoglobin is key. Roggan et al carried out optical studies on whole blood, which showed an absorption minimum at 805 nm for fully de-oxygenated whole blood with maximas at 420 and 540 nm.10,11 This confirms that ECL emission in this region of the NIR spectra will suffer significantly less quenching from whole blood compared to emission in the visible region.

Therefore, NIR QDs are promising materials for incorporation into ECL biosensors with detection directly from whole blood. Investigations into the ECL behavior and performance of these QDs in whole blood have not been carried out previously, to the best of our knowledge. It has been shown that NIR QDs can generate a strong ECL signal in both the cathodic and anodic regions. To date, the study of NIR emitters for biosensing applications has focused on work under ideal conditions in buffer, which whilst achieving optimal performance does not take advantage of the key property of these materials, namely their emission in the NIR region. As a result, there is a clear requirement for investigations into the ECL properties of these materials and examinations into the behavior of NIR emitters under conditions in which their unique properties over visible region emitters are truly scrutinized to confirm the suitability of these materials for use in biological samples, such as whole blood. Dopamine is a catecholamine involved in a wide array of physiological processes. It acts within the brain and central nervous system (CNS) as a neurotransmitter and disruption within these systems is linked to a variety of diseases including schizophrenia,12 depression,13 and Parkinson’s disease.14 Dopamine is not solely involved in the CNS, but also acts on the immune system where it helps to dictate the activity of effector cells,15 the cardiovascular system where it influences factors such as blood pressure and contraction of heart muscles,16 and the renal system where it is known to influence blood supply to the kidney and production of urine.17 Detection of dopamine is clearly of clinical significance and there are a number of techniques used to do so including voltammetry,18,19 fluorescence,20 and ECL.21,22 Detection via ECL is based upon quenching of the response through an energy-

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transfer process23 and dopamine was therefore selected as an ideal candidate for use in these investigations into whole blood ECL detection as it is often used as a model analyte in ECL investigations but has never been examined in blood. In this work, we have developed a biosensor for dopamine based on the ECL of 800 nm CdSeTe/ZnS core-shell QDs. A glassy carbon electrode (GCE) was modified with a QD/chitosan composite. Chitosan was selected as the polymer for film development due to its non-toxicity, good biocompatibility and commercial availability.22,23 This work has shown that these NIR-emitting QDs are suitable for use in ECL biosensors in complex matrices such as whole blood, which has not previously been examined, and could be extremely helpful in the development of novel systems that are able to detect clinically relevant analytes directly from clinical samples. They have been used to successfully develop a dopamine detection system with a clinically-relevant linear range, minimizing the requirement for sample preparation. Ideally, this system could be used within a multiplexed platform and this is an area in which future work would focus.

EXPERIMENTAL Instrumentation To elucidate the suitability of NIR QDs for dopamine detection, electrochemical measurements were carried out using a CH instrument model 760D electrochemical analyzer. All experiments were carried out using a conventional threeelectrode assembly, consisting of a 3 mm diameter GC working electrode, Pt wire counter electrode and Ag/AgCl reference electrode. GC electrodes were cleaned by successive polishing using 1, 0.3 and 0.05 µM alumina slurry, followed by sonication in ethanol and water, respectively, for 30 mins. The electrodes were then dried under a flow of N2 gas. Cyclic voltammetry (CV) was carried out at a scan rate of 100 mV s1 and sample interval of 1 mV across a potential range outlined in each figure. For differential pulse voltammetry (DPV), the increment potential was 4 mV, amplitude 50 mV, pulse width 50 ms, sample width 16.7 ms and pulse period 0.5 s across a potential range outlined in each figure. Measurements involving simultaneous detection of ECL signals and current utilized a CH instrument model 760D connected to a Hamamatsu H10723-20 PMT. The PMT was biased by +5 V. The scan rate was 100 mVs-1. During electrochemical experiments, the cell was kept in a light-tight Faraday cage in a specially designed holder configuration where the working electrode was positioned directly above the PMT window. All measurements were made at room temperature. Materials Core-shell CdSeTe/ZnSQDs (Qdot® 800 ITK™ organic quantum dots, 1 µM in decane) were purchased from Invitrogen. Core-shell CdSe/ZnS QDs (Lumidot™ 560 and 640 nm QDs, 5 mg/mL in toluene), chitosan (medium molecular weight, 75-85% deacetylated), phosphate buffered saline (PBS, pH 7.4), potassium persulfate (K2S2O8), dopamine hydrochloride were all purchased from Sigma-Aldrich and used as received. All other reagents used were of analytical grade, and all solutions were prepared in milli-Q water (18 mΩ cm). Bovine whole blood was obtained from Wishaw Abattoir Ltd (185 Caledonian Road, Wishaw, Lanarkshire, ML2 0HT)

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and stored in aliquots at -20 ºC. Aliquots were defrosted at room temperature on the day of analysis and used immediately.

METHODS Preparation of CdSeTe/ZnS core-shell QD-chitosan films To fabricate the NIR QD composite films in chitosan, the CdSeTe/ZnS core-shell QDs were first converted to a water soluble form. This was done following the method developed by Woelfle and Claus6. 0.5 mL of 0.5 M DAET in methanol was mixed with 0.25 mL of the CdSeTe/ZnS QDs in decane (1 µM) / CdSe/ZnS QDs in toluene (5 mg/mL). Nitrogen was bubbled through the solution for 5 mins, which was then sealed and left stirring overnight at room temperature in the dark. The QDs were then precipitated with an excess of acetone followed by centrifugation at 5000 rpm for 6 mins. The filtrate was removed and the precipitate was re-dispersed in 0.25 mL of distilled water. These water-soluble QDs were centrifuged for a further 6 mins at 3000 rpm to remove any impurities and then stored in darkness in the fridge. The QD/chitosan composite was then prepared by mixing aliquots of the water-soluble QDs with the chitosan solution in a 1:1 (v/v) ratio. 3 µL of this composite was then carefully cast onto the electroactive portion of a GC electrode and allowed to dry in the fridge for 1 h in the dark. A film of bare QDs was prepared in the same manner, with water used instead of chitosan. Whole blood samples Whole blood samples were diluted with 0.1 M PBS in a 9:1 (v/v) ratio. Whole blood samples containing K2S2O8 were diluted with 10 mM K2S2O8 in a 9:1 (v/v) ratio to obtain a working concentration of 1 mM K2S2O8. Dopamine samples For the analysis of dopamine, a 1 mM stock solution was prepared in 0.1 M PBS and diluted to the required concentration in 0.1 M PBS. For blood samples containing dopamine, 0.038, 0.075, 0.375, 0.75, 1.5, 2, 3 and 4.5 mM stock solutions of dopamine were prepared in 0.1 M PBS and diluted 1:9 (v/v) in whole blood to obtain final dopamine working concentrations of 3.75, 7.5, 37.5, 75, 150, 200, 300 and 450 µM.

RESULTS AND DISCUSSION The electrochemical behavior of NIR QDs in whole blood was investigated via cyclic voltammetry. Figure 1 shows this electrochemical behavior in the anodic region. The blank GC electrode voltammogram exhibits a single, irreversible oxidation peak at 0.55 V. This peak is not present with a blank GC in phosphate buffered saline (PBS) and so is related to oxidation of a whole blood species. Albumin is one of the most abundant proteins in blood, having a concentration of approximately 40 mg/mL.24 It can exist in its oxidized or reduced form, with oxidation occurring via a free thiol group on the amino acid cysteine.25 The oxidation of cysteine has been reported on a bare GC electrode at similar potentials26 indicating this peak is likely caused by oxidation of albumin in the whole blood sample. This is further confirmed as an increase in peak current of this process was also observed following QD/chitosan film modification of the GC electrode26 as is seen in Figure 1. In addition a poorly defined oxidation peak at ~1.2

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V is apparent for the QD/chitosan film is observed. Peak current at this potential is dependent upon QD concentration and is therefore related to an oxidation process involving the QDs themselves. This is more clearly evident from the differential pulse voltammogram (DPV) shown in Figure 2. 800

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Figure 1. Typical cyclic voltammetric response of blank GC electrode (black dashed line) and GC electrode modified with 800 nm QD/chitosan film (red solid line) in whole blood at a scan rate of 100 mV s-1 over the potential range 0 ≤ ν ≤ 2 V vs. Ag/AgCl. 70 60

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have an influence of the electrochemical properties of the QD/chitosan film. Typically ECL based biosensors rely on co-reaction systems,27 however within this study we will focus our examination of co-reactant free systems. The co-reactant free system with NIR QDs is known to produce a strong signal through interaction between QDs(h+1Sh) and superoxide.23,28 The ECL profile of QD/chitosan film in whole blood is shown in Figure 3. An ECL emission associated with the oxidation peak at 1.1 V is observed after electrode modification with the QD/chitosan film, indicating the response is a result of emission from the QDs. The similarity of anodic ECL profiles in PBS, (see Supplementary Figure S1), and whole blood confirm that the same electron transfer processes are taking place, which involves electron transfer between O2- and oxidized QDs formed during the potential scan. It appears that no significant quenching of ECL precursors by a species in whole blood is occurring as the magnitude of ECL response is comparable to that in PBS. The significantly larger signal observed in the anodic co-reactant free system compared to previous cathodic systems29 may be due to the faster route for formation of O2-, which requires a single electron transfer step compared to several for OH• formation. Therefore, the concentration of OH• is likely to be lower as there are a number of steps available for quenching events. This data shows that intense anodic NIR QD ECL can be produced in whole blood without the need for any additional co-reactants, which can permit development of novel co-reactant free systems with direct blood analysis. The key advantage of these QDs over visible region QD ECL emitters is their NIR emission.

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Figure 2. Typical DPV current response of blank GC electrode (black dashed line) and 800 nm QD/chitosan film (red solid line) in whole blood at a scan rate of 100 mV s-1 over the potential range 0 ≤ ν ≤ 2 V vs. Ag/AgCl.

The bare GC electrode exhibits an oxidation peak at 0.45 V, consistent with oxidation of albumin as seen in the voltammograms from CV. The improved sensitivity and slower scan rates associated with DPV allow observation of two defined oxidation peaks not seen in the voltammetric profiles from CV. These are centered at 1.0 V and 1.55 V respectively and are seen following electrode immobilization of the QD/chitosan film, in both whole blood (Figure 2). These processes are associated with hole injection into different energy levels of the QDs electronic structure. This data indicates that the anodic electrochemical behavior of NIR QDs in whole blood is similar to that observed in in PBS as no additional oxidation peaks related to QD processes are observed and therefore this more complex media does not

ECL Intensity / A.U.

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Figure 3. ECL response of blank GC electrode (black dashed line) and 800 nm QD/chitosan film (red solid line) in whole blood at a scan rate of 100 mV s-1 over the potential range 0 ≤ ν ≤ 2 V vs. Ag/AgCl monitored simultaneously with CV data shown in Figure1.

A comparison of the detectable ECL response from 800, 640 and 560 nm QDs was carried out to investigate the penetrability of this light signal through whole blood. A strong emission from the co-reactant free system in whole blood meant this method was used for comparison. However, it was discovered that 560 and 640 nm QDs could not produce a detectable ECL signal with this system, as the redox potential of the O2-/O2 couple were not be sufficient to inject an electron

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Analytical Chemistry into the LUMO levels of these QDs (which will be higher than the LUMO level of 800 nm QDs owing to their smaller size and larger Eg). Therefore, tripropylamine (TPA) was used for this comparison as all investigated QDs had the ability to generate an ECL signal with this co-reactant. Figure 4 shows the result of these comparisons and Table 1 the maximum ECL intensities and potentials at which they occur. In the potential region examined, only the 800 nm NIR QDs exhibit a detectable ECL signal, which shows that light generated by 640 and 560 nm QDs in whole blood spiked with 20 mM TPA do not have sufficient intensity and penetrability to reach the detector. With 800 nm QDs, a strong oxidative ECL peak at 1.2 V is observed, which provided further confirmation that these QDs are ideal for use as ECL emitters directly from whole blood. This data has shown that NIR QD ECL has significantly improved penetrability through whole blood samples when compared to QDs with emission in the visible region. This is the first time such a comparison has been made and exemplifies the excellent optical properties associated with NIR QDs for use with direct blood analysis.

800 nm QD 640 nm QD 560 nm QD

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anodic ECL response with O2-. Therefore, these NIR QDs are unique in their ability to both generate a strong anodic ECL response without an additional co-reactant and do so at such a wavelength that interference directly from whole blood is minimal. To highlight the application of these NIR QDs for bioanalytical detection in whole blood, we utilized this system to detect dopamine within whole blood samples. The effect of dopamine concentration on the ECL response of NIR QDs in PBS was examined first and is shown in Figure 5. The profile exhibits an oxidative ECL peak at 1.20 V, which decreases in intensity in response to increasing dopamine concentration. The relationship between the quenching of this ECL response and the dopamine concentration was examined utilizing the Stern-Volmer equation (Eq. 1). In order for efficient quenching to occur, the acceptor and donor molecules must be in close contact so that their electron clouds can interact.30,31 This quenching can be static, which relates to the formation of a ground state donor-acceptor complex or dynamic, which is diffusion controlled and involves collisions between donor and acceptor molecules, preventing emission and the Stern-Volmer equation can be used to evaluate this and to calculate the quenching constant for this system.30,31 = 1 + (1) where I0 is the initial ECL intensity, I the ECL intensity at a specific concentration of quencher [Q], and KSV is the SternVolmer quenching constant.

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Figure 4. Typical ECL responses for 800 nm (black line), 640 nm (red line) and 560 nm (blue line) QD/chitosan film in whole blood spiked with 20 mM TPA at a scan rate of 100 mV s-1 over the potential range 0.5 ≤ ν≤ 2 V vs. Ag/AgCl.


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0 3.75 7.5 37.5 75 100 150 200 300 450

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[Dopamine] / µM

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Table 1. Ep and ECL intensities of 800, 640 and 560 nm QDs in whole blood spiked with 20 mM TPA. QD Emission λ / nm 800 640 560

Ep V vs. Ag/AgCL 1.20 n/a n/a

ECL Intensity A.U. 1569 n/a n/a

Overall, the results shown highlight that NIR QDs can be used to generate anodic ECL in whole blood without the requirement for an additional co-reactant. The significance of this lies in the fact that an extremely simple system can be used for generation of an intense ECL response from whole blood. This is very attractive for development of biosensors (and particularly POC devices) as it minimizes cost, sample preparation and any disruption to a biological system that may occur following co-reactant addition. This work indicates that only QDs with a sufficiently small band gap can generate an

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Figure 5. ECL response of 800 nm QD/chitosan film in 0.1 M PBS containing 0 ≤ [dopamine] ≤ 450 µM at a scan rate of 100 mV s-1 over the potential range 0 ≤ ν≤ 2.0 V vs. Ag/AgCl. Inset shows the maximum ECL response of 800 nm QD/chitosan film in these solutions at ~1.20 V vs. Ag/AgCl. Error bars are shown for n = 3 but are smaller than the size of the data points.

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y = 231.94x + 0.5521 R² = 0.9971

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1 / [Dopamine] / µM

Figure 6. Modified Stern-Volmer plot for 800 nm QD/chitosan film in (red circles) 0.1 M PBS and in whole blood (blue squares) with increasing concentrations of dopamine from 0 to 450 µM.

For both static and dynamic quenching, this predicts a linear relationship between Io/I and [Q] and thus a plot of these parameters has a slope that is equal to KSV. However, a SternVolmer plot of data from Figure 5 does not exhibit this linear dependence and instead requires a modification to the SternVolmer equation, Figure 6. Previous investigations have shown that the quenching process can be influenced if the emitting species is confined within a polymer film.32-34 The so–called Hindered Access Model is a two state model that takes into account the fact that some species are accessible and others inaccessible to the quencher. Here, this is caused by confinement of the QDs within the chitosan film, where some species are accessible for quenching and others are isolated from quencher molecules. This modification to the SternVolmer equation (Eq 2) predicts a linear relationship between (I0/I0 - I) and (1/[Q]), which yields an intercept equal to fa-1 and a slope of faKa-1.31 1 1 = + (2) − Where fa is the fraction of accessible species and Ka is the Stern-Volmer quenching constant of this accessible fraction. Using this modified model, a plot of the data generates a linear relationship, suggesting that the chitosan polymer is influencing the quenching behavior in the system as shown in Figure 6. This yields a linear range of 3.75 to 450 µM dopamine and a Ka of 1.8 x 104 M-1s-1, which indicates that dopamine, is an effective quencher of NIR QD ECL emission. This quenching is a result of an energy transfer process between the excited state of the QDs and the oxidation product of dopamine, οbenzoquinone.23 This oxidation product can also be utilized as a confirmatory signal for the presence of dopamine within the sample. The voltammograms (Figure 7) exhibit a peak at 0.35 V, which is attributed to the oxidation of dopamine to οbenzoquinone as its peak potential (inset, Figure 7) is dependent upon dopamine concentration. This can be utilized as a secondary or confirmatory check for the presence of dopamine within a sample matrix.

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Figure 7. Current response of 800 nm QD/chitosan film in whole blood spiked with 37.5 (black), 75 (red), 150 (blue), 200 (pink), 300 (green) and 450 µM (navy) dopamine at a scan rate of 100 mV s-1 over the potential range 0 ≤ ν ≤ 2 V vs. Ag/AgCl. Inset is the linear dependence of this peak current at 0.35 V vs. Ag/AgCl on dopamine concentration.

Collision between this species and excited state QDs results in quenching of the ECL signal, permitting detection of dopamine. As dopamine could effectively quench the ECL of NIR QDs in buffer, detection from whole blood was attempted. Figure 8 shows the effect of dopamine concentration on the ECL response of the QD/chitosan film in whole blood. The profile exhibits an ECL signal at 1.15 V that is linearly dependent on the concentration of dopamine in the whole blood sample over the range 0.375 - 450 µM, similar to that observed in PBS solution (Figure 5). The modified Stern-Volmer plot (Figure 6) yielded a Ka value of 1.5 x 103 M-1s-1. This rate is approximately nine times lower than that achieved in buffer, which indicates that de-activation of ο-benzoquinone QDs* quenching ability is occurring, likely through energy transfer with another species in blood that is in the vicinity of the electrode surface. This calibration in whole blood (Figures 6 and 8) shows good linearity and based on the signal to noise (S/N) of 3, the detection limit is estimated to be 100 nM. Analysis of the secondary peak at 0.35 V can also be utilized to verify this limit and/or the presence of dopamine in whole blood samples. Alternative ECL biosensors also have dopamine linear ranges in the µM range.21,22 However, the significance of this biosensor is that ECL detection of dopamine was achieved directly from whole blood and no additional co-reactants were required in the system. It is the first time such a system has been developed and provides further confidence in the ability of these QDs to be effective and responsive ECL emitters in whole blood.

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Table 2. Average calculated concentrations and recoveries from unknown whole blood serum samples.

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37.5 75 150

39 71.8 137.5

HPLC Averaged Calc. Conc. (µ (µM) 37.35 76.2 149.8

% Recoveries

104.42 94.23 91.92

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Dop 100 uM 5 mM 10 mM 100 uM 5 mM 10 mM 100 uM 5 mM 10 mM

Interferent

Figure 9. Dependence of the ECL response at 1.15 V for the 800 nm QD/chitosan film in whole blood (WB) with 37.5 µM dopamine (Dop), ascorbic acid (blue), citric acid (purple) and uric acid (green) at 100 µM, 2 mM and 4 mM respectively. Error bars represent triplicate data points.

Selectivity is a very important aspect of sensor performance. Thus, commonly existing species such as ascorbic acid, citric acid and uric acid are chosen to evaluate the selectivity of the as-fabricated NIR QD ECL based dopamine sensor. Therefore the clinical viability and cross-reactivity of this system was highlighted and the influence of these commonly encountered interferents often found in clinical blood samples (glucose, urea, citric acid) on the ECL response of this biosensor is shown in Figure 9. None of these had any appreciable effect on the observed ECL intensity indicating good sensor specificity. Within the current system, these species did produce an ECL response but that response was at a higher oxidation potential than that observed for dopamine (see Supplementary Figure S2). This is similar to other reports which highlighted the versatility of ECL detection for selective detection strategies, although the influence of the electrolyte pH is extremely important.35-38 If the pH is altered this can often cause issues and/or improvements in selectivity towards a given analyte.37 Indeed the percentage recoveries for these samples was found to be ≤± 5% when compared to chromatographic analysis as shown in Table 2. A typical response from the chromatographic analysis is shown in the supplementary material (see Supplementary Figure S3).

The response from multiple biosensors was investigated to determine the reproducibility of this system and is shown in Figure 10 and over a three week period was examined to give an indication of the shelf-life of this biosensor. A consistent ECL response from the QDs is apparent over the considered period, with variation in intensity of only 5.2% after three weeks. Response variability increases at the three week point, indicating biosensor reproducibility may suffer after this time. Co-reactant free anodic ECL of the NIR QDs in blood exhibits an intense oxidation peak at 1.10 V, whereas 640 and 560 nm QDs generate no such detectable response. Anodic ECL was responsive to the addition of reactive molecules, such as dopamine, even in this complex matrix. Within this work a co-reactant free anodic ECL biosensor for dopamine was developed and exhibited good linearity in the mM and µM range respectively. Although the NIR QDs give lower ECL intensities compared to other electrochemical systems, this is compensated for by the reduction in sample preparation, and subsequent advantages of requiring fewer reagents and the capability to detect dopamine in this complex biological matrix. Future work will focus on enhancement strategies to improve this limit of detection to facilitate detection in biologically relevant concentration ranges. 6800


Figure 8. ECL response of 800 nm QD/chitosan film to increasing concentrations of dopamine spiked in whole blood. The range analyzed was 0 ≤ [dopamine] ≤ 450 µM dopamine at a scan rate of 100 mV s-1 over the potential range 0 ≤ ν≤ 1.9 V vs. Ag/AgCl.


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Figure 10. Average ECL response for 800 nm QD/chitosan film on day 1, after 24 hours, after 1 week and after 3 weeks. Error bars represent standard deviations for n = 6.

CONCLUSION Significantly, this is the first ECL biosensors to be developed with detection directly from whole blood and have demonstrated the capability and versatility of NIR QD ECL as an option for the development of innovative biosensors with a particular focus on POC detection. It highlights the capacity of

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ECL biosensors to effectively detect a medically relevant biomarker in whole blood without the need for sample pretreatment or extraction procedures to be undertaken. Future work would is being undertaken to enhance the sensitivities and to allow for multiplexed biomarker recognition.

ASSOCIATED CONTENT Supporting Information. ECL response of a GC electrode modified with 800 nm QD/chitosan film in 0.1 M PBS, the ECL response for a 800 nm QD/chitosan film in 0.1 M PBS containing 100 mM dopamine and 2 mM ascorbic acid and a typical chromatogram for the detection of dopamineare given. Chromatographic experimental details are also included. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected], Tel: +44 (0)141 548 4322; Fax: +44 (0)141 548 2532

Author Contributions The manuscript was written through contributions of all authors.

Funding Sources This work was supported by funding from the EU FP7 funding through the Marie Curie Reintegration Grant scheme (PIRG2010-268236).

Notes Any additional relevant notes should be placed here.

ACKNOWLEDGMENT This material is based upon works supported by the EU FP7 Marie Curie Reintegration Grant No. PIRG-2010-268236.

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Whole Blood Electrochemiluminescent Detection of Dopamine

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