Quantum dots as chemiluminescence enhancers tested by sequential injection technique: Comparison of flow and flow-batch conditions

Author’s Accepted Manuscript Quantum Dots as chemiluminescence enhancers tested by sequential injection technique: Comparison of flow and flow-batch conditions Hana Sklenářová, Ivona Chocholouš, Miroslav Polášek

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S0022-2313(16)30992-9 http://dx.doi.org/10.1016/j.jlumin.2016.12.030 LUMIN14445

To appear in: Journal of Luminescence Received date: 27 July 2016 Revised date: 21 December 2016 Accepted date: 22 December 2016 Cite this article as: Hana Sklenářová, Ivona Voráčová, Petr Chocholouš and Miroslav Polášek, Quantum Dots as chemiluminescence enhancers tested by sequential injection technique: Comparison of flow and flow-batch conditions, Journal of Luminescence, http://dx.doi.org/10.1016/j.jlumin.2016.12.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Quantum Dots as chemiluminescence enhancers tested by sequential injection technique: Comparison of flow and flow-batch conditions

Hana Sklenářová1, Ivona Voráčová2, Petr Chocholouš1, Miroslav Polášek1

1

Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of

Analytical Chemistry, Hradec Králové, Czech Republic 2

Institute of Analytical Chemistry of the CAS, v. v. i., Brno, Czech Republic

Corresponding author: Hana Sklenářová, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Analytical Chemistry, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic, [email protected]

Abstract The effect of 0.01 – 100 µmol L-1 Quantum Dots (QDs) with different emission wavelengths (520 – 640 nm) and different surface modifications (mercaptopropionic, mercaptoundecanoic, thioglycolic acids and mercaptoethylamine) on permanganate-induced and luminol–hydrogen peroxide chemiluminescence (CL) was studied in detail by a sequential injection technique using a spiral detection flow cell and a flow-batch detection cell operated in flow and stopflow modes. In permanganate CL system no significant enhancement of the CL signal was observed while for the luminol–hydrogen peroxide CL substantial increase (> 100% and > 90% with the spiral detection cell in flow and stop-flow modes, respectively) was attained for CdTe QDs. Enhancement exceeding 120% was observed for QDs with emissions at 520, 575 and 603 nm (sizes of 2.8 nm, 3.3 nm and 3.6 nm) using the flow-batch detection cell in the stop-flow mode. Pronounced effect was noted for surface modifications while mercaptoethylamine was the most efficient in CL enhancement compared to mercaptopropionic acid the most commonly applied coating. Significant difference between results obtained in flow and flow1

batch conditions based on the entire kinetics of the extremely fast CL reaction was discussed. The increase of the CL signal was always accompanied by reduced lifetime of the CL emission thus application of QDs in flow techniques should be always coupled with the study of the CL lifetime.

Keywords Quantum Dots; chemiluminescence; sequential injection analysis

1. Introduction Quantum dots (QDs) exhibit special optical properties that could be applied in fluorescence as well as in chemiluminescence (CL) analytical assays. According to previous research [1-5] enhanced or amplified CL signal was explained as a result of catalysis of redox reactions or charge transfer based on chemiluminescence resonance energy transfer (CRET, involving nonradiative (dipole–dipole) transfer of energy from a chemiluminescent donor to a suitable acceptor molecule) principle or combination of both. Catalytic properties were defined as size- and shape-dependent [1] and their effect was observed in batch and flow systems as well. The enhancement of the CL emission was applied for trace analysis in different areas including immunochemistry, biochemistry and bio-imaging [2,3]. Catalysis of CL reactions was exploited in relation to CdTe QDs in luminol-hydrogen peroxide CL system where QDs accelerated electron-transfer process leading to a more intense CL emission was achieved [3-5]. Principle of the CRET was described for the overlap between the CL emission spectrum and absorption spectrum of fluorescent acceptor [3], e.g. CRET between luminol (donor) and QDs (acceptor). Advantages of CRET against fluorescence resonance energy transfer (FRET, involving a donor fluorophore in an excited electronic state, which may transfer its excitation energy to a nearby acceptor chromophore) are mainly based on following parameters: no auto-fluorescence background signal, no photobleaching, broad excitation spectra, large Stokes shifts, high quantum yield, photo-stability and size-dependent emission wavelength tunability [2]. Testing of QDs properties and also their applications for sensitive determination of different analytes was often carried out in automated flow systems [6-8]. Under flow conditions excellent repeatability was combined with increased selectivity, reproducible reaction control, 2

versatile monitoring, high sample throughput and sufficient sensitivity due to the increased CL signal even though measured in non-steady state conditions [6]. Solubility of QDs could be increased by surface modifications. In most publications mercaptopropionic acid (MPA) or thioglycolic acid (TGA) [6,9] were applied with high stability of aqueous dispersions of the modified QDs and the surface modification could be also connected with the CL enhancement. As proposed in the previously published literature [6] the CL enhancement in direct permanganate-induced CL system is connected with catalytic properties of QDs and proportional relation between the CL signal and QDs size. On the other hand, the CL enhancement in the luminol-H2O2 system was found to be based mostly on the CRET mechanism (where the relation between the CL intensity and QDs size was inversely proportional). The CRET mechanism was proved previously by overlapped emission spectra of luminol-CdTe QDs conjugates [10] and in the study of luminol-H2O2 CL with core-shell QDs (ZnS/CdSe) [11]. The aim of this work was to study in detail the effect of QDs with different coatings on permanganate-induced and on luminol-hydrogen peroxide CL systems by the sequential injection analysis (SIA) technique with detection cells of different designs (a flow and a flowbatch cell) to compare both approaches with respect to extremely fast kinetics of the CL reactions. The two CL systems were chosen in order to study the effect of QDs on the CL signal in red and blue emission regions, in fast and longer-lasting CL signal and direct catalysis and CRET mechanism, respectively. Relation between the CL enhancement and QDs size and the effect strength of QDs coating on the lifetime of the CL signal were also examined. The indirect luminol-hydrogen peroxide system was selected as the most commonly used CL system for sensitive determination of different inorganic ions capable of catalytic properties Cu(II), Co(II), Cr(III), Fe(II), Fe(III), Zn(II), Cd(II), Al(III) - with the perspective to study potential of the CL enhancement for determination of these ions after post-column derivatization using ion-exchange low-pressure chromatography [12] and it is intended to be applied for screening in trace metals analysis. Novelty of the work is based on flow and flow-batch geometries applied for comparison of both size and concentration dependent QDs influence. The observed effect was found to be closely related to the time of the chemiluminescence signal scanning while the enhancement 3

effect was always connected with the signal duration shortening. Different coating of the QDs surface commonly used for regular dispersion in aqueous conditions was not tested with respect to the potential chemiluminescence enhancement till now and corresponded to high difference not only in the CL intensity and in the signal lifetime too.

2. Experimental 2.1.

Chemicals

Ultra-pure water produced by Milli-Q (Millipore, USA) system was used for preparation of all solutions throughout the study. Tested CdTe QD nanoparticles were prepared in the Institute of Analytical Chemistry of the CAS, v. v. i., Brno, Czech Republic. The QDs coated with mercaptopropionic acid, exhibiting emission maxima at 520, 575, 603 and 636 nm (MPA520, MPA575, MPA603 and MPA636) and sizes of 2.8 nm, 3.3 nm, 3.6 nm and 4.0 nm, respectively, were tested. In the second part of the study, where the effect of the QDs surface modification of CdTe or CdTe/CdS nanoparticles was examined, the following QDs were employed: CdTe QDs coated with mercaptoundecanoic acid (emission at 520 nm, size 2.8 nm, MUA520), with mercaptopropionic acid (540 nm; 3.0 nm, MPA540), with thioglycolic acid (620 nm; 3.8 nm TGA620) or with mercaptoethylamine (590 nm; 3.5 nm, MEA590) and additionally the CdTe/CdS particles coated with mercaptopropionic acid (640 nm; 4.0 nm, MPA640) [13,14] for comparison with MPA coated CdTe nanoparticles. Sizes of QDs were determined using dependence of luminescence emission maxima on CdTe core size. Molar concentrations of CdTe QDs were determined from approximate molar mass of QDs core. This molar mass was calculated using CdTe core size and crystal structure [14]. Hydrophilic CdTe quantum dots were prepared by a two-step reaction described elsewhere [13,14]. Briefly, sodium hydrogen telluride was prepared by the reaction of NaBH4 and Te powder in a flask with degassed water, cooled by water with ice cubes and stirred for approximately 6 hours. Next, the dark violet solution of NaHTe was injected into the reaction bottle with degassed solution of cadmium chloride and a thiolated ligand in water. Thiolated ligands, MPA, MUA, TGA, were used for the preparation of negatively charged QDs and MEA in case of positively charged QDs. The molar ratio Cd2+: HTe- : ligand was 2 : 1 : 4.8. Finally, the pH was adjusted to 9.5 – 10.0 in case of MPA, TGA and MUA or 5 in case of 4

MEA. The reaction mixture was heated under the reflux condenser for specified time (several hours) to reach the desired size of nanocrystals. CdS coated QDs were prepared by reaction of CdTe quantum dots coated with MPA and CdCl2, Na2S and MPA. The whole suspension was refluxed for about one hour. Phloroglucinol from AnalaR (England), hydroquinone, pyrocatechol and sulfuric acid from Lachema (Czech Republic), gallic acid from Sigma (Germany), resorcinol from Penta (Czech Republic) and potassium permanganate from Balex (Czech Republic) of p.a. quality were used in permanganate-induced CL experiments. The experiments with the luminol CL system were carried out with luminol from Fluka (Czech Republic), hydrogen peroxide (30%, v/v) from Sigma-Aldrich (Czech Republic), potassium hexacyanoferrate(III) from Balex (Czech Republic) and sodium hydroxide from Penta (Czech Republic).

2.2.

Apparatus

The sequential injection (SIA) setup (see Fig. 1) was a lab-designed flow system consisting of a piston pump Cavro XL-3000 (2.5 mL), a 10-port selection valve Vici Valco, a holding coil made of PTFE tubing (volume of 1.2 mL, i.d. 0.75 mm) and a computer with PCIe 6251 card for data acquisition (National Instruments) and PCI 232.2 (Netmos) card for the communication with the SIA system. A FaFSIA lab-made software based on LabVIEW® programming language was used for the SIA system control and data treatment. The CL detection cells were of two different measurement designs. The first one was a common spiral flow cell made of transparent PTFE tubing (0.5 mm i.d.), see Fig. 1a. The spiral was fixed on Perspex plate mounted in the PMT-based fluorimetric detector (Schoeffel Instruments, Germany) placed instead of an emission filter. The working voltage of the PMT was 250 V. Both photomultipliers scan data with the frequency of 10 Hz. The flow-batch CL detection device was a lab-made Perspex cell with transparent bottom (volume 1 mL) seated on the window of the Hamamatsu low-voltage photosensor module (H5784-01, Hamamatsu Photonics K. K., Japan) operated at the voltage of 4 V. The cell involved 3 channels allowing the aspiration of the reactants, for emptying the cell, and ventilation (Fig. 1b).

5

Data scanning frequency for both detection systems was 10 Hz, in the stop-flow mode the CL signal was monitored for the period of 60 s.

Figure 1

2.3.

Measurement procedure

Optimization of the aspiration zone sequence, volumes of reagents and even their concentrations and flow rates was not included but it was based on our previous experience with the same flow system, detection cells and photomultipliers – volumes were kept in the same ratio, concentrations were optimized with respect to the working voltage of both detectors, zone sequence and flow rates were optimal for efficient mixing and quick transport of the reacting zones to the detector. All measurements were carried out in triplicate and the mean values were used for the evaluation and comparison of the effect of the studied QD nanoparticles together with the repeatability expressed as RSD (%). For data evaluation the peak height in flow measurements and the peak area for stop-flow measurements were considered.

2.3.1. Flow measurement Measurements with the spiral flow cell involved aspiration of all zones into the holding coil followed by their injection into the spiral flow cell (while the CL signal was recorded and evaluated as the CL peak height) and then to waste. The volume of each reacting zone was 50 µL and the flow-rate of their transport to the detection cell was 100 µL s-1 (quick transport towards the detector was needed). Proper mixing of all reacting zones was accomplished by aspiration of additional 70 µL of water and one flow reversal between the aspiration and detection steps. This flow scheme was used in QD experiments with either permanganate or luminol CL systems. In the case of permanganate system, the sequence of aspiration was: 50 µL of QDs, 50 µL of 2.5 mmol L-1 analyte (gallic acid, pyrocatechol, hydroquinone, resorcinol, phloroglucinol), 50 µL of 0.25 mol L-1 sulfuric acid, and 50 µL of 5 mmol L-1 potassium permanganate. QDs were tested at 4 concentration levels prepared from an aqueous stock solution with 6

concentration of 0.1 mmol L-1: 10, 1, 0.5 and 0.1 µmol L-1. All measurements were corrected with blank CL signal obtained by aspirating a zone of water instead of the tested QDs solution. In the case of luminol CL system the aspiration sequence was: 50 µL of 1 mmol L-1 hydrogen peroxide, 50 µL of 1 mmol L-1 potassium hexacyanoferrate(III), 50 µL of 1 mmol L-1 luminol in 0.1 mol L-1 NaOH and 50 µL of QDs in the following concentration levels (25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025 and 0.01 µmol L-1). Correction for the blank signal was carried out the same way as in the permanganate system. Additionally, stop-flow measurements were carried out by stopping the reacting zones in the spiral cell and recording the CL signal for 60 s.

2.3.2. Flow-batch measurement The flow-batch system involved aspiration of the reactant zones separated by air bubbles (to prevent contact of the reactants prior to detection) into the flow-batch detection cell. Immediate mixing of the reactants inside the cell allowed monitoring of the kinetics of the CL reaction from the very beginning that is the main difference from flow measurements; the CL signal was recorded for 60 s. The sequence of aspiration was: 10 µL of air, 40 µL of 1 mmol L-1 luminol in 0.1 mol L-1 NaOH, 10 µL of air, 10 µL of 20 mmol L-1 potassium hexacyanoferrate(III), 10 µL of air, 40 µL of 0.01 mol L-1 hydrogen peroxide, 10 µL of air and 50 µL of QDs with concentration of 100, 10, 1 µmol L-1. Higher concentrations of potassium hexacyanoferrate(III) and hydrogen peroxide were used to cover the whole scale of the low-voltage photosensor and thus to modify the measurement because of lower sensitivity compared to high-voltage photomultiplier used in the previous system.

3. Results and discussion 3.1.

Permanganate-induced chemiluminescence under flow conditions

In this part of the study five different analytes from the group of polyphenols that could be easily oxidized by permanganate in acidic medium (to produce excited Mn(II) species that emit CL [13]) were tested under flow conditions because of the fast reaction and short lifetime 7

of the CL signal. Thus only flow set-up with spiral detection cell (and high-voltage photomultiplier because of lower intensity of the CL signal) was applied and evaluation of the fast CL signal based on the peak height was carried out. Only slight enhancement of the CL signal in the presence of MPA520, MPA575, MPA603 and MPA636 compared to the blank signal was observed. The evaluation was based on the percentage of the increase/decrease of the original CL signal (blank) and the respective values are documented in Table 1 where values exceeding 10% were highlighted in bold. The CL enhancement of MPA520 was more pronounced compared to the effect of larger nanoparticles. As for the concentration effect of QDs lower concentrations of MPA520 (1 – 0.1 µmol L-1) increased the CL signal by more than 10% in the reaction of permanganate with 3 analytes (gallic acid, resorcinol and phloroglucinol). In the oxidation of gallic acid also MPA575 in the lowest concentration (0.1 µmol L-1) caused the increase of CL signal. In case of pyrocatechol the CL signal was affected only slightly. The effect on the CL signal accompanying the oxidation of hydroquinone CL was ambiguous; at different QDs concentrations enhancement from  23% to suppression of 17% was observed. Repeatability of CL signal (n = 3) in the experiments with MPA520, MPA575, MPA630 and MPA636 was characterized by RSD values ranging from 0.20 to 3.50% when values exceeding 1.50% were only rare. This indicates that QD suspensions behave as solutions in the flow system without the risk of precipitation on the tubing walls or in the flow cell.

Table 1

Generally, no distinctive enhancement or suppression of the CL signal in the permanganateinduced CL emission was observed in this part of the study. The relation of the CL signal on the QDs size was reported earlier [1,6] and it was proved in our experiments too. The mechanism of the signal enhancement in the permanganate-induced CL was expected to be based on the catalysis of the redox reaction with decrease of the CL signal in case of larger QDs that was previously observed in direct QDs oxidation [6,15] but also combined with the emission of Mn(II) species obtained in reduction of permanganate [16]. Low effect of QDs under the flow conditions did not allow to deduce the main principle easily but combination of both mechanisms could be proposed (catalysis and QDs oxidation). 8

3.2.

Luminol chemiluminescence

Effect of QDs on the luminol-hydrogen peroxide CL was tested with both flow and flowbatch detection arrangements. Because of more intense luminol CL, both a high-voltage and a low-voltage photomultiplier could be applied.

3.2.1. The flow-batch detection arrangement (low –voltage) In the system with flow-batch detector a peak height and a peak area were evaluated by monitoring the change of the CL signal for 60 s after mixing the reactants. The same QDs as in the permanganate-induced CL (MPA520, MPA575, MPA603 and MPA636) were tested in the concentration range of 100 – 1 µmol L-1. In the luminol-hydrogen peroxide system more pronounced differences in the CL signal enhancement by different QDs were observed compared to the permanganate-induced CL. The results are summarized in the Table 2 where enhancements exceeding 100% are highlighted in bold. For higher concentration levels of QDs a quenching in the range of approximately 38 – 100% was observed throughout all emission wavelengths except of MPA520 when considering the CL peak height. For QD concentrations of 10 and 1 µmol L-1 the CL signal increased by ≈ 33 to 129%, respectively. Significant increase was found for the lower concentration levels in case of MPA520, MPA575 and MPA603. For QD concentrations of 100 and 10 µmol L-1 noticeable decrease of CL peak areas and shorter duration of the CL emission was demonstrated. The RSD values (n = 6) of the CL peak heights and CL peak areas for lower concentrations of QDs were 2.5 – 6.0% and 3.9 – 7.0%, respectively. The repeatability was worsened in experiments with the highest QDs concentration (0.1 mmol L-1).

Table 2

The observed QDs size and concentration dependent CL enhancement/inhibition in the luminol-hydrogen peroxide system was in good agreement with the previously published findings [1,6]. Effect of the size of QDs was inversely related to the extent of the CL enhancement. The enhancement efficiency was found to be inversely proportional to the 9

concentration of QDs as well; 1 µmol L-1 QDs (the lowest concentration tested) were more efficient compared to higher concentrated QDs. When evaluating the CL peak 10 – 1 µmol L-1 QDs caused the CL enhancement while the highest concentration level of QDs (100 µmol L-1) reduced the CL signal. In case of enhancement evaluation based on the peak area measurement (monitoring the change of the CL intensity in time) 100 – 10 µmol L-1 QDs shortened the lifetime of CL while lower concentrations of QDs slowed down the CL decay. Hence the expected mechanism of the CL enhancement was confirmed to be based on the CRET [6] and inverse relation between the QDs size and CL emission was proved in accordance with the previously published results of CL enhancement in the luminol-hydrogen peroxide system with mercaptosuccinic acid (MSA) coated CdTe QDs [17]. The kinetics that was observed in the flow system with injection valve placed close to the detector [17] was applied to differentiate CL signal o IgG bio-conjugates and corresponded with our fast reaction scanning in flow-batch conditions.

3.2.2. The flow detection arrangement with the spiral cell (high-voltage) Here the same experimental conditions except of concentrations of hydrogen peroxide and potassium hexacyanoferrate (both 1 mmol L-1) were used. Again the peak height and the peak area (with 60 s stop-flow period) were evaluated. The results are shown in Table 3. Decrease of the CL intensity and shortening of the CL signal were observed when examining the effect of MPA575, MPA603 and MPA636 (the effect of QDs decreased with decreasing concentration). The smallest QDs (MPA520) did cause significant differences in terms of the CL enhancement compared to the blank experiments at lower QD concentration levels. The RSD values of the CL signal under the flow conditions ranged from 0.11 to 5.44% and again higher RSD values were found at higher QDs concentration. The CL enhancement effect of QDs was not noted for any size of the QDs. Higher QD concentration caused more pronounced CL inhibition. Comparison of the obtained results testing the same QDs in the same concentration levels showed significant difference between both approaches (flow and flow-batch) that is documented in Fig. 2. Small enhancement or even inhibition of the CL signal under flow conditions could be caused by different CL kinetics of the QDs tested. While flow-batch system allows to follow the CL reaction from the very beginning, common flow system with mixing and transfer to the detector failed in

10

scanning the fast CL signal increase and hence the QDs enhancement effect could not be observed.

Table 3 Figure 2

3.3.

Study of the effect of QDs coating

This part of the study was focused on the comparison of the effect of different coatings of QDs. The study was carried out in the flow system with spiral detection cell within the QDs concentration range of 25 – 0.01 µmol L-1. The concentration of QDs causing the highest increase of the CL signal was used for the stop-flow measurement. The results are documented in Fig. 3 and Fig. 4. The raw data are summarized in supplementary Table S1 where the most significant CL enhancement results are highlighted in bold in the upper part of the Table S1. The results of the stop-flow experiments with the MEA-QDs are highlighted as the most efficient with respect to enhancement of the CL signal intensity and reduction of the CL lifetime (see the lower part of the Table S1).

Figure 3 Figure 4

Higher increase of the CL signal ( 57 – 105%) was found for QDs with lower emission wavelengths – MUA520, MPA540 and MEA590 – all in higher concentration levels (10 µmol L-1). QDs with higher emission wavelengths - TGA620, MPA636 and MPA640 – did not affect the CL signal so efficiently, the increase ranged from  8 to 35%. The core-shell CdTe/CdS nanoparticles (MPA640) compared to MPA636 revealed the CL enhancement 2 – 10-times higher and shifted to lower concentration levels (0.1 µmol L-1). The MEA590 QDs were found to be the most effective for the enhancement of the CL signal in the luminol – H2O2 system causing the highest increase of the CL signal intensity ( 91%) and at the same time considerable reduction of the CL lifetime while the peak area decreased by 76%. The 11

real records of the MEA590 QDs effect are documented in Fig. 5, where again different kinetics was observed even under the flow conditions.

Figure 5

Also here the CRET was found to be the main principle of the CL enhancement due to the observed proportional relation between the CL signal intensity and concentration of the QDs (except of the core-shell CdTe/CdS nanoparticles). The form of coating affected the CL signal significantly even in very quick measurements under flow conditions. The best efficiency in the CL signal enhancement was achieved with the MEA-coated QDs that was proved additionally in stop-flow experiment. The highest increase of the CL signal was accompanied by dramatic shortening of the CL signal in time. Such kinetics of the CL signal was mentioned previously in the paper dealing with the influence of metal ions on the CdTe/H2O2 chemiluminescence where the effect of QDs of different sizes on the CL kinetics was studied [18]. Such a short lifetime of emission in luminol-hydrogen peroxide CL system was not expected while variability of the CL kinetics with QDs surface modifiers was not studied and discussed before. Explanation could be found in the transfer of energy from the original CL system to QDs and thus kinetics of the emitted signal corresponds to QDs emission that is different from the emission in the CL system. Speed of the CL reactions can cause substantial problems with the QDs-CL applications in flow systems where enhancement was mainly expected to be caused by stabilization of the CL signal [6,7].

4. Conclusion Comprehensive testing of potential enhancement of the CL by QDs in direct (permanganateinduced) and indirect (luminol – H2O2) systems using flow and flow-batch conditions including stop-flow measurements was carried out. In the CL reactions of permanganate with different polyphenols only slight effect of MPA-QDs based on CdTe was observed for smallest particles with emission at 520 nm under flow conditions (direct transport of the reactants to the spiral detection cell). 12

The effect of QDs on the luminol-H2O2 CL system was studied under the flow and the flowbatch conditions (with spiral and flow-batch detection cells); more distinct enhancement was observed in the flow-batch system at lower concentrations of all tested QDs. The increase of the CL signal reached 120%.

The most significant effect of the QDs surface modifiers was observed with the mercaptoethylamine coating; such QDs caused the increase of the CL signal intensity by more than 100% while the original lifetime of CL was reduced to 25% (monitored for 60 s in the stop-flow mode). To compare the effect of QDs size, concentration level and coating tested under flow conditions different surface modifications revealed higher difference of the CL signal in terms of potential enhancement. The results of the study in the luminol-H2O2 can be summarized as: (i) the CL enhancement was achieved in flow-batch SIA arrangement and partially in conventional flow SIA system; (ii) the CL enhancement was confirmed for different emission wavelengths of QD nanoparticles; (iii) the CL enhancement was strongly dependent on the concentration of QDs; and (iv) the extent of the CL enhancement was significantly different for QDs with different surface coatings. Differentiation between direct catalysis of CL reaction by QDs and CRET mechanism was not so straightforward if compared with findings published earlier [6]. In both CL systems inversely proportional relation between the CL enhancement and the QDs size was found which indicates possible CRET mechanism (in luminol-hydrogen peroxide system) and combination of catalysis by QDs with CRET (in permanganate-induced CL). From the practical point of view, the tested QD nanoparticles affected the CL signal only in limited concentration range and the enhancement effect was significantly dependent on the QD size and surface modification. The enhancement of the CL signal was often not prominent and it was strongly dependent on all experimental conditions including the type of CL reaction, arrangement of the flow system and the detection mode. In view of these facts application of QDs as CL enhancers in flow systems require thorough optimization of a number of experimental parameters (including proper selection of the type, surface modification of the QDs and geometry of the flow system for the scanning very fast CL reactions).

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Acknowledgement The authors gratefully acknowledge financial support of the Ministry of Education, Youth and Sports of the Czech Republic, project VES13 Kontakt II LH13023.

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Figure captions

Fig. 1: Scheme of the sequential injection system with (a) spiral and (b) flow-batch detection cells Fig. 2: Comparison of the QDs effect on the luminol-hydrogen peroxide CL emission in flow (F) and flow-batch (FB) conditions (evaluated by peak height) Fig. 3: The effect of QDs coating and QDs concentration on the enhancement of CL in the luminol–hydrogen peroxide reaction – flow measurements Fig. 4: Survey of extreme values of the CL enhancement and suppression in the luminol– hydrogen peroxide reaction for different types of QDs; CL enhancement evaluated by the peak height; CL suppression evaluated by the peak area; for details see supplementary Table S1 Fig. 5: Real records of the CL signal in the stop-flow measurement with MEA-coated QDs; comparison of (a) blank luminol – H2O2 reaction (1 mmol L-1 luminol in 0.1 mol L-1 NaOH, 1 mmol L-1 hexacyannoferate(III) and 1 mmol L-1 H2O2) and (b) the same as (a) but + 10 µmol L-1 MEA-coated 590 nm QDs

Table 1: The effect of the type and concentration of QDs on the percentage increase/decrease of permanganate-induced chemiluminescence signal intensity – flow measurements

Analyte

Gallic acid

Pyrocatechol

QD conc.

QD type/emission wavelength [nm]

[µmol L-1]

MPA/520

MPA/575

MPA/603

MPA/636

10.0

7.6

3.2

-6.5

-1.6

1.0

10.8

0.9

-4.2

-1.8

0.5

26.7

0.2

-2.5

-2.3

0.1

17.1

17.6

-2.5

2.3

10.0

1.1

2.2

-0.3

-1.4

1.0

0.6

2.2

0.6

-1.1

16

Resorcinol

Hydroquinone

Phloroglucinol

0.5

-0.8

3.3

4.4

0.6

0.1

-4.6

-0.6

-2.2

-3.0

10.0

4.7

3.4

5.8

1.9

1.0

14.8

4.3

7.1

3.9

0.5

14.2

8.6

3.0

5.0

0.1

12.0

-3.9

0.0

0.9

10.0

4.6

1.3

1.9

1.1

1.0

8.2

1.9

-12.6

3.2

0.5

22.9

1.9

3.2

1.3

0.1

2.3

-17.2

-6.5

-7.1

10.0

9.8

0.8

-1.9

0.0

1.0

12.7

3.3

-2.8

-0.6

0.5

15.0

3.3

0.2

-0.2

0.1

10.2

-2.3

-1.5

-3.7

MPA – mercaptopropionic acid

Table 2: The effect of the type and concentration of QDs on the percentage increase/decrease of CL signal in the luminol - hydrogen peroxide reaction – flow-batch measurements

Parameter

QD conc.

QD type/emission wavelength [nm]

evaluated

[µmol L-1]

MPA/520

Peak height

100.0

Peak area

MPA/575

MPA/603

MPA/636

22.2

-37.8

-53.3

-100.0

10.0

44.4

37.8

35.6

33.3

1.0

128.9

120.0

122.2

48.9

100.0

-53.9

-77.0

-96.1

-100.0

10.0

-43.9

-44.7

-48.9

-50.3

1.0

57.4

55.4

53.3

-33.8

MPA – mercaptopropionic acid

17

Table 3: The effect of the type and concentration of QDs on the percentage increase/decrease of CL signal in the luminol - hydrogen peroxide reaction – flow measurements

Parameter

QD conc.

QD type/emission wavelength [nm]

evaluated

[µmol L-1]

MPA/520

MPA/575

MPA/603

MPA/636

Peak height

100.0

-36.3

-73.4

-70.9

-72.5

10.0

1.1

-45.6

-40.4

-51.8

1.0

3.2

-17.2

-12.6

-17.7

100.0

-35.5

-73.0

-71.4

-72.6

10.0

1.3

-45.6

-41.7

-52.4

1.0

4.5

-18.5

-7.9

-18.3

Peak area

MPA – mercaptopropionic acid

18

19

20

Graphical abstract

21

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