Lab-on-Microsphere—FRET-Based Multiplex Sensor Platform

Here we report on the development and investigation of a novel multiplex assay model based on polymer microspheres (PMS) encoded with ternary AIS/ZnS quantum dots (QDs). The system was prepared via layer-by-layer deposition technique. Our studies of Förster resonance energy transfer (FRET) between the QD-encoded microspheres and two different cyanine dyes have demonstrated that the QD photoluminescence (PL) quenching steadily increases with a decrease in the QD-dye distance. We have found that the sensitized dye PL intensity demonstrates a clear maximum at two double layers of polyelectrolytes between QDs and Dye molecules on the polymer microspheres. Time resolved PL measurements have shown that the PL lifetime decreases for the QDs and increases for the dyes due to FRET. The designed system makes it possible to record spectrally different bands of FRET-induced dye luminescence with different decay times and thereby allows for the multiplexing by wavelength and photoluminescence lifetimes of the dyes. We believe that PMS encoded with AIS/ZnS QDs have great potential for the development of new highly selective and sensitive sensor systems for multiplex analysis to detect cell lysates and body fluids’ representative biomarkers.


Introduction
Quantum dots (QDs) are semiconductor nanoparticles in which the motion of electrons is limited in all three spatial dimensions. QDs are well-known for tuneable photoluminescence (PL) bands [1][2][3] broad absorption spectra [1,4], great photostability in comparison to the organic dyes [5] and high PL quantum yields [6]. These unique characteristics have made them particularly interesting for both fundamental research and commercial applications [7,8]. QDs have already been applied in the fields of photonics and biomedicine, including systems for energy conversion, light-emitting devices, bioimaging tools, sensing platforms, and, since recently, assays for the multiplex analysis [9]. Optoelectronic and spectroscopic properties of the QDs strongly depend on their chemical composition, size, shape and surface chemistry [10,11], which can be controlled by implementing diverse synthetic strategies and modification techniques [12][13][14].
Quantum dots are generally synthesized of semiconductor binary compounds such as II-IV and IV-VI elements [15]. The most well-known examples include Cd-and Pbbased QDs. These QDs have been extensively investigated in the last few decades and remain prospective materials due to the well-studied chemical and optical properties and simple manufacturing techniques [16,17] However, cadmium and lead toxicity hinders further application of these quantum dots in biomedicine and, considering the latest environmental regulations, in optoelectronic technologies [18,19] The non-toxic ternary

Synthesis of AIS/ZnS QDs
Hydrophilic synthesis of AgInS 2 /ZnS quantum dots was performed according to the previously described procedure [47,48]. This approach is based on the controllable reaction between sodium sulfide, silver (I) and indium (III) mercaptoacetate complexes. Briefly, AgNO 3 was loaded in a three-necked round bottom flask filled with water under continuous stirring. After addition of TGA, the solution turned turbid. Then, the resulting solution turned yellow and transparent after adding NH 4 OH aqueous solution. Next, InCl 3 in HNO 3 0.2 M aqueous solution was injected, that led to discoloration of the mixture because of the mercaptoacetate complexes formation. Then, Na 2 S solution was added and the solution became orange. After that the flask was heated up to 95 • C for 30 min with further addition of TGA, Zn(CH 3 COO) 2 solution and NH 4 OH solutions for ZnS shell growth. Obtained mixture was one more time heated up to 95 • C for 30 min. At the end of the reaction, the flask was cooled down and the solution was rotary evaporated and further purified by size-selective precipitation using i-PrOH. The fracture of AIS/ZnS QDs of yellow color was redissolved in Millipore water for further use.

Preparation of AIS/ZnS-Doped Microspheres
One milliliter of a 0.5 M NaCl solution of a poly(allylamine hydrochloride) (PAH) polyelectrolyte with the concentration of 5 mg/mL was added to the precipitated polystyrene microspheres. The resulting dispersion was shaken for 10 min. To remove excess PAH polyelectrolyte, the solution was centrifuged for 30 s at 1500 rcf, the supernatant was removed and the precipitate was washed with distilled water. Then 1 mL of 0.5 M NaCl solution of a poly(sodium 4-styrenesulfonate) (PSS) polyelectrolyte with a concentration of 5 mg/mL was added to the coated spheres. The resulting dispersion was shaken for 10 min to remove an excess of PSS polyelectrolyte and then the solution was centrifuged for 30 s at 4000 rpm, the supernatant was removed and the precipitate was washed with distilled water. This polyelectrolyte coating and purification procedure was repeated until five PAH/PSS binary layers were formed. After the obtained ten-layer coating with polyelectrolytes, a positive PAH polyelectrolyte layer was formed on the surface by the similar method described above.
Then, 200 µL of a concentrated aqueous solution of AIS/ZnS QDs was added to the precipitated microspheres. The resulting dispersion was shaken overnight. To remove unreacted QDs, the solution was centrifuged for 30 s at 4000 rpm, the supernatant was removed and the precipitate was washed with distilled water. The resulting spheres with an outer layer of QDs are additionally covered with different numbers of PAH/PSS binary layers (1-5) by a similar method described for the previous layers (samples L1-L5).
Cy3 and Cy5 dyes were added to each sample with 1-5 PAH/PSS binary layers, samples were shaken for 5 min and washed 2 times with distilled water. The concentration of PMS and dyes slightly varied from sample to sample after washing procedure, which was determined from UV-Vis spectra. All PL spectra of the samples were corrected for the concentration.

Equipment
The UV-VIS absorption spectra were recorded using a UV-Probe 3600 spectrophotometer (Shimadzu, Kyoto, Japan). The steady-state PL spectra were obtained with Cary Eclipse spectrofluorometer (Agilent, Santa Clara, CA, USA). Time-resolved fluorescence spectroscopy measurements were performed using a time-correlated single photon counting (TCSPC) fluorescence microscope MicroTime 100 (PicoQuant, Inc. Inc., Berlin, Germany) equipped with a 405 nm pulsed laser LDH-P-C-405B (PicoQuant) with pulse duration of 20 ps. The signal was collected by a single photon photomultiplier tube detector; different detection wavelengths were selected with a holographic bandpass filter with the 10 nm bandwidth tunable in the spectral range of 430-780 nm. Laser frequency was adjusted using an additional signal generator SFG-71003 (Good Will Instek, Montclair, CA, USA). The fluorescent images were obtained with confocal laser scanning microscope LSM 710 (Carl Zeiss, Oberkochen, Germany) based on upright stand Axio Imager Z1 with objective EC Epiplan-Apochromat 50×/0.95. For excitation luminescence was used diode laser with 405 nm wavelength. PL signal was collected by the 32-channel spectral detector QUASAR.

Spectroscopy of AIS QDs and Cyanine Dyes
The normalized PL spectra of AgInS 2 /ZnS QDs (AIS QDs) and UV-Vis and PL spectra of Cy3 and Cy5 dyes are presented in Figure 1. The PL band maximum of AIS is 555 nm, full width at half maximum is 121 nm. The PL quantum yield is 38.6%. The quantum yield was estimated relatively against rhodamine 6G. The PL bands of Cy3 and Cy5 dyes have maxima at 570 nm and 663 nm, respectively. The donor (QD) PL and acceptors (Cy3 or Cy5) absorption bands overlap well, providing a possibility to FRET from QDs to the molecules. In our experiments, to facilitate the observation and analysis of the energy transfer from QD to dyes, PL of AgInS 2 /ZnS QDs was excited at 350 nm, where the absorption of dyes is negligible, while Cy3 and Cy5 were irradiated at 500 nm and 600 nm respectively since at these wavelengths the QDs do not absorb.

Equipment
The UV-VIS absorption spectra were recorded using a UV-Probe 3600 spectrophotometer (Shimadzu, Kyoto, Japan). The steady-state PL spectra were obtained with Cary Eclipse spectrofluorometer (Agilent, Santa Clara, CA, USA). Time-resolved fluorescence spectroscopy measurements were performed using a time-correlated single photon counting (TCSPC) fluorescence microscope MicroTime 100 (PicoQuant, Inc. Inc., Berlin, Germany) equipped with a 405 nm pulsed laser LDH-P-C-405B (PicoQuant) with pulse duration of 20 ps. The signal was collected by a single photon photomultiplier tube detector; different detection wavelengths were selected with a holographic bandpass filter with the 10 nm bandwidth tunable in the spectral range of 430-780 nm. Laser frequency was adjusted using an additional signal generator SFG-71003 (Good Will Instek, Montclair, CA, USA). The fluorescent images were obtained with confocal laser scanning microscope LSM 710 (Carl Zeiss, Oberkochen, Germany) based on upright stand Axio Imager Z1 with objective EC Epiplan-Apochromat 50×/0.95. For excitation luminescence was used diode laser with 405 nm wavelength. PL signal was collected by the 32-channel spectral detector QUASAR.

Spectroscopy of AIS QDs and Cyanine Dyes
The normalized PL spectra of AgInS2/ZnS QDs (AIS QDs) and UV-Vis and PL spectra of Cy3 and Cy5 dyes are presented in Figure 1. The PL band maximum of AIS is 555 nm, full width at half maximum is 121 nm. The PL quantum yield is 38.6%. The quantum yield was estimated relatively against rhodamine 6G. The PL bands of Cy3 and Cy5 dyes have maxima at 570 nm and 663 nm, respectively. The donor (QD) PL and acceptors (Cy3 or Cy5) absorption bands overlap well, providing a possibility to FRET from QDs to the molecules. In our experiments, to facilitate the observation and analysis of the energy transfer from QD to dyes, PL of AgInS2/ZnS QDs was excited at 350 nm, where the absorption of dyes is negligible, while Cy3 and Cy5 were irradiated at 500 nm and 600 nm respectively since at these wavelengths the QDs do not absorb.

Confocal and Fluorescence Lifetime Imaging of PMS-AIS and PMS-AIS-Dyes
As a typical example, confocal PL and transmission images of microspheres with QDs on the surface (PMS-AIS) as well as with QDs and Cy5 dye (PMS-AIS-Cy5) are shown in Figure 2. The PL image of PMS-AIS was registered with an excitation wavelength of 405 nm and emission wavelength region of 450-600 nm (Figure 2a). QDs cover the PMS surface quite evenly but in some regions bright spots are observed, probably associated   Figure 2f represents the superposition of the QD and Cy5 PL. The bright spots of Cy5 sensitized PL correspond to QD bright PL areas as FRET occurs more intensively in these regions.  Figure 2f represents the superposition of the QD and Cy5 PL. The bright spots of Cy5 sensitized PL correspond to QD bright PL areas as FRET occurs more intensively in these regions.

UV-Vis Spectroscopy of PMS-AIS and PMS-AIS-Dyes
Microspheres have strong scattering due to the relatively large particle size, which is reflected in their extinction spectra as an appearance of a very high background ( Figure  3). Absorption spectra of dyes comprised of PMS-AIS-dye complexes were obtained by subtraction of the background. Figure 3 demonstrates, as an example, the absorption spectra of Cy5 dye in the PMS-AIS-Cy5 complexes, which has two absorption bands: 595 nm and 647 nm. The peak at 647 nm is also observed in the absorption spectrum of Cy5 in a free state and corresponds to the monomeric form. The peak at 595 nm corresponds to the Cy5 dimer [28] and its appearance indicates a partial aggregation of the dye upon adsorption on PMS-AIS.

UV-Vis Spectroscopy of PMS-AIS and PMS-AIS-Dyes
Microspheres have strong scattering due to the relatively large particle size, which is reflected in their extinction spectra as an appearance of a very high background ( Figure 3). Absorption spectra of dyes comprised of PMS-AIS-dye complexes were obtained by subtraction of the background. Figure 3 demonstrates, as an example, the absorption spectra of Cy5 dye in the PMS-AIS-Cy5 complexes, which has two absorption bands: 595 nm and 647 nm. The peak at 647 nm is also observed in the absorption spectrum of Cy5 in a free state and corresponds to the monomeric form. The peak at 595 nm corresponds to the Cy5 dimer [28] and its appearance indicates a partial aggregation of the dye upon adsorption on PMS-AIS.  where QDs absorb the light, while the dye does not have absorption and, therefore, cannot be directly excited. As a result of FRET, the QD PL is quenched and the dye sensitized PL appears. For clarity, the PL spectra of PMS-AIS-dyes with two double spacer layers of polyelectrolytes (L2) are deconvoluted by gaussian AIS and Cy3 PL bands. To address the distance dependence of the photoexcitation energy transfer from QDs to dyes, we studied the quenching of the QD luminescence and the change in the intensity of the sensitized PL of dyes as a function of the number of polyelectrolyte layers. The thickness of one double PAH/PSS layer is approximately 1 nm [49], consequently, 5 layers are approximately 5 nm. Calculated FRET radii of the QD-Cy3 and QD-Cy5 pairs are 5.39 and 6.67 nm, respectively. We found that the degree of PL quenching increases monotonically with a decrease in the number of layers and QD PL is quenched most intensively for PMS-AISdyes without any and with one double layer of polyelectrolytes (1L). It is reasonable, since, in addition to FRET, charge carrier transfer, usually responsible for the most effective QD PL quenching process, prevails at 1-2 nm distance between a QD and a dye molecule. The sensitized PL of the dyes also drops with the number of layers since the efficiency of FRET decreases with distance. However, for the PMS-AIS-dyes, the sensitized PL intensity possesses a noticeable maximum at L2. This is most likely due to the appearance of an additional nonradiative energy relaxation channel when the distance between QDs and dye molecules becomes close enough for electron transfer from QDs to dyes. Thus, the samples with 2-3 polymer layers have optimal conditions for FRET, because the distance between the QDs and the dyes is less than the Förster radius and the FRET efficiency is high. Further, for samples L4 and L5, the FRET efficiency decreases.  Calculated FRET radii of the QD-Cy3 and QD-Cy5 pairs are 5.39 and 6.67 nm, respectively. We found that the degree of PL quenching increases monotonically with a decrease in the number of layers and QD PL is quenched most intensively for PMS-AIS-dyes without any and with one double layer of polyelectrolytes (1L). It is reasonable, since, in addition to FRET, charge carrier transfer, usually responsible for the most effective QD PL quenching process, prevails at 1-2 nm distance between a QD and a dye molecule. The sensitized PL of the dyes also drops with the number of layers since the efficiency of FRET decreases with distance. However, for the PMS-AIS-dyes, the sensitized PL intensity possesses a noticeable maximum at L2. This is most likely due to the appearance of an additional nonradiative energy relaxation channel when the distance between QDs and dye molecules becomes close enough for electron transfer from QDs to dyes. Thus, the samples with 2-3 polymer layers have optimal conditions for FRET, because the distance between the QDs and the dyes is less than the Förster radius and the FRET efficiency is high. Further, for samples L4 and L5, the FRET efficiency decreases.   Figure 4 represents PL spectra of PMS-AIS and PMS-AIS-dye excited at 350 nm, where QDs absorb the light, while the dye does not have absorption and, therefore, cannot be directly excited. As a result of FRET, the QD PL is quenched and the dye sensitized PL appears. For clarity, the PL spectra of PMS-AIS-dyes with two double spacer layers of polyelectrolytes (L2) are deconvoluted by gaussian AIS and Cy3 PL bands. To address the distance dependence of the photoexcitation energy transfer from QDs to dyes, we studied the quenching of the QD luminescence and the change in the intensity of the sensitized PL of dyes as a function of the number of polyelectrolyte layers. The thickness of one double PAH/PSS layer is approximately 1 nm [49], consequently, 5 layers are approximately 5 nm. Calculated FRET radii of the QD-Cy3 and QD-Cy5 pairs are 5.39 and 6.67 nm, respectively. We found that the degree of PL quenching increases monotonically with a decrease in the number of layers and QD PL is quenched most intensively for PMS-AISdyes without any and with one double layer of polyelectrolytes (1L). It is reasonable, since, in addition to FRET, charge carrier transfer, usually responsible for the most effective QD PL quenching process, prevails at 1-2 nm distance between a QD and a dye molecule. The sensitized PL of the dyes also drops with the number of layers since the efficiency of FRET decreases with distance. However, for the PMS-AIS-dyes, the sensitized PL intensity possesses a noticeable maximum at L2. This is most likely due to the appearance of an additional nonradiative energy relaxation channel when the distance between QDs and dye molecules becomes close enough for electron transfer from QDs to dyes. Thus, the samples with 2-3 polymer layers have optimal conditions for FRET, because the distance between the QDs and the dyes is less than the Förster radius and the FRET efficiency is high. Further, for samples L4 and L5, the FRET efficiency decreases. (c) (d)

PL Spectra of PMS-AIS and PMS-AIS-Dyes Excited at 520 and 600 nm
Since in the present system, there is a potential competition between the sensitized PL of the dyes and quenching of the luminescence of dyes due to the electron transfer, which have different distance dependences, we studied the effect of the polyelectrolyte spacer layer thickness on the intensity of the dye intrinsic PL in the PMS-AIS-dye. The intrinsic PL spectra of Cy3 and Cy5 excited at 520 and 600 nm, respectively, where QDs do not absorb light, are shown in Figure 5. Spectra were corrected to dye concentration. A noticeable quenching of the dye PL is observed for L1 and L2, being the most pronounced for L1, which confirms the assumption of additional nonradiative channel formation in the vicinity of the QDs. The luminescence of the samples L3-L5 is approximately at the same level.

Time-Resolved PL Measurements of PMS-AIS and PMS-AIS-Dyes
For the implementation of TR-FRET, it is important that the luminescence lifetime of AIS QDs depends on the wavelength of the detected radiation. The AIS QDs PL decay curves measured at different detection wavelengths were well fitted by two exponents:

PL Spectra of PMS-AIS and PMS-AIS-Dyes Excited at 520 and 600 nm
Since in the present system, there is a potential competition between the sensitized PL of the dyes and quenching of the luminescence of dyes due to the electron transfer, which have different distance dependences, we studied the effect of the polyelectrolyte spacer layer thickness on the intensity of the dye intrinsic PL in the PMS-AIS-dye. The intrinsic PL spectra of Cy3 and Cy5 excited at 520 and 600 nm, respectively, where QDs do not absorb light, are shown in Figure 5. Spectra were corrected to dye concentration. A noticeable quenching of the dye PL is observed for L1 and L2, being the most pronounced for L1, which confirms the assumption of additional nonradiative channel formation in the vicinity of the QDs. The luminescence of the samples L3-L5 is approximately at the same level.

PL Spectra of PMS-AIS and PMS-AIS-Dyes Excited at 520 and 600 nm
Since in the present system, there is a potential competition between the sensitized PL of the dyes and quenching of the luminescence of dyes due to the electron transfer, which have different distance dependences, we studied the effect of the polyelectrolyte spacer layer thickness on the intensity of the dye intrinsic PL in the PMS-AIS-dye. The intrinsic PL spectra of Cy3 and Cy5 excited at 520 and 600 nm, respectively, where QDs do not absorb light, are shown in Figure 5. Spectra were corrected to dye concentration. A noticeable quenching of the dye PL is observed for L1 and L2, being the most pronounced for L1, which confirms the assumption of additional nonradiative channel formation in the vicinity of the QDs. The luminescence of the samples L3-L5 is approximately at the same level.

Time-Resolved PL Measurements of PMS-AIS and PMS-AIS-Dyes
For the implementation of TR-FRET, it is important that the luminescence lifetime of AIS QDs depends on the wavelength of the detected radiation. The AIS QDs PL decay curves measured at different detection wavelengths were well fitted by two exponents:

Time-Resolved PL Measurements of PMS-AIS and PMS-AIS-Dyes
For the implementation of TR-FRET, it is important that the luminescence lifetime of AIS QDs depends on the wavelength of the detected radiation. The AIS QDs PL decay curves measured at different detection wavelengths were well fitted by two exponents: The average PL lifetimes was calculated by the following equation: where A 1 and A 2 are the amplitudes and τ 1 and τ 2 are the decay times of the first and second exponents, respectively. The values of these parameters are shown in Table 1. The absorption bands of Cy3 and Cy5 overlap different spectral regions of the AIS QD PL band having different lifetimes. Due to FRET, the PL lifetime decreases for QDs and increases for dyes ( Figure 6). The average PL lifetimes was calculated by the following equation: where and are the amplitudes and and are the decay times of the first and second exponents, respectively. The values of these parameters are shown in Table 1. The absorption bands of Cy3 and Cy5 overlap different spectral regions of the AIS QD PL band having different lifetimes. Due to FRET, the PL lifetime decreases for QDs and increases for dyes ( Figure 6). Intrinsic PL lifetimes of Cy3 and Cy5 are approximately 0.6 and 0.5 ns, respectively. In complexes with QDs the PL lifetime of Cy3 and Cy5 increased to 20 and 30 ns at the maxima of their PL bands 580 and 665 nm, respectively ( Table 1). The AIS QDs have PL lifetimes increasing with PL excitation wavelengths, as was shown previously [50]. Since Cy3 has an absorption band overlapping with the QD PL band at lower wavelengths with shorter lifetimes than Cy5, the lifetime of sensitized PL of Cy3 is also smaller, which can be used in time-resolved multiplexing.  Intrinsic PL lifetimes of Cy3 and Cy5 are approximately 0.6 and 0.5 ns, respectively. In complexes with QDs the PL lifetime of Cy3 and Cy5 increased to 20 and 30 ns at the maxima of their PL bands 580 and 665 nm, respectively ( Table 1). The AIS QDs have PL lifetimes increasing with PL excitation wavelengths, as was shown previously [50]. Since Cy3 has an absorption band overlapping with the QD PL band at lower wavelengths with shorter lifetimes than Cy5, the lifetime of sensitized PL of Cy3 is also smaller, which can be used in time-resolved multiplexing. Figure 7 presents the FLIM of PMS-AIS, PMS-AIS-Cy3 and PMS-AIS-Cy5 measured at maxima of corresponding PL bands using the 10 nm bandpass filters, demonstrating the significant change in the lifetimes at the wavelength of sensitized dye PL. These images illustrate the ability of the developed PMS-AIS-dyes system for multiplexed sensing with conventional bandpass spectral techniques and time selection techniques.

Conclusions
In this paper, we have developed a multiplex assay model that is based on polymer microspheres encoded with AIS/ZnS QDs and investigated their interaction with two different cyanine dyes emitting in different spectral regions. Our studies have shown that the QD PL quenching steadily increases with the decrease in the number of polyelectrolyte layers between the QDs and the dyes. As a result, the QD PL quenches the most for PMS-AIS-dyes with no or just one double layer of polyelectrolytes. However, the sensitized dye PL intensity demonstrates a clear maximum in the case of two double layers in PMS-AISdyes, which has been attributed to the presence of an additional nonradiative energy relaxation channel when the distance between QDs and dye molecules. In addition, direct excitation of the dyes has demonstrated a visible quenching for the samples with one and two double layers of coating confirming the presence of an additional nonradiative channel in the vicinity of the QDs. Time-resolved PL measurements of PMS-AIS and PMS-AISdyes systems have shown that the PL lifetime decreases for QDs and increases for dyes because of FRET. PL lifetimes of AIS QDs also increases with the PL excitation wavelength and Cy3 dye's absorption overlaps with the QD PL band at lower wavelengths with shorter lifetimes than those for Cy5 dye. The study clearly demonstrates the potential of the proposed model to record the spectrally separated bands of FRET-induced luminescence for dyes with different decay times. Therefore, these PMS-based systems can be used to achieve multiplexing by wavelength and photoluminescence lifetimes of the dyes. We also believe that PMS encoded with AIS/ZnS QDs have a potential for design of highly selective and sensitive sensors for multiplex analysis to detect cell lysates and body fluids' representative biomarkers.

Institutional Review Board Statement:
The study did not involve humans or animals.

Informed Consent Statement:
The study did not involve humans.

Data Availability Statement:
The study does not report data which present the ethical, legal or privacy issues. The data can be shared.

Conclusions
In this paper, we have developed a multiplex assay model that is based on polymer microspheres encoded with AIS/ZnS QDs and investigated their interaction with two different cyanine dyes emitting in different spectral regions. Our studies have shown that the QD PL quenching steadily increases with the decrease in the number of polyelectrolyte layers between the QDs and the dyes. As a result, the QD PL quenches the most for PMS-AIS-dyes with no or just one double layer of polyelectrolytes. However, the sensitized dye PL intensity demonstrates a clear maximum in the case of two double layers in PMS-AIS-dyes, which has been attributed to the presence of an additional nonradiative energy relaxation channel when the distance between QDs and dye molecules. In addition, direct excitation of the dyes has demonstrated a visible quenching for the samples with one and two double layers of coating confirming the presence of an additional nonradiative channel in the vicinity of the QDs. Time-resolved PL measurements of PMS-AIS and PMS-AIS-dyes systems have shown that the PL lifetime decreases for QDs and increases for dyes because of FRET. PL lifetimes of AIS QDs also increases with the PL excitation wavelength and Cy3 dye's absorption overlaps with the QD PL band at lower wavelengths with shorter lifetimes than those for Cy5 dye. The study clearly demonstrates the potential of the proposed model to record the spectrally separated bands of FRET-induced luminescence for dyes with different decay times. Therefore, these PMS-based systems can be used to achieve multiplexing by wavelength and photoluminescence lifetimes of the dyes. We also believe that PMS encoded with AIS/ZnS QDs have a potential for design of highly selective and sensitive sensors for multiplex analysis to detect cell lysates and body fluids' representative biomarkers.

Institutional Review Board Statement:
The study did not involve humans or animals.

Informed Consent Statement:
The study did not involve humans.

Data Availability Statement:
The study does not report data which present the ethical, legal or privacy issues. The data can be shared.