ORIGINAL PAPER

A lysosome-targeting nanosensor for simultaneous fluorometricimaging of intracellular pH values and temperature

Wei Zhang1& Y. G. Abou El-Reash1

& Longjiang Ding1& Zhenzhen Lin1

& Ying Lian1& Bo Song2

& Jingli Yuan2&

Xu-dong Wang1

Received: 14 September 2018 /Accepted: 1 October 2018# Springer-Verlag GmbH Austria, part of Springer Nature 2018

AbstractLysosomal pH and temperature are two crucial physiological parameters that are involved in regulating intracellular homeostasis,and their precise measurements are extremely important in understanding this process and diseases diagnosis. A lysosome-targeting nanosensor has been designed for simultaneous imaging of pH values and temperature in HeLa cells. Three dyes werecovalently immobilized either inside or on silica nanoparticles. The nanosensors have an average diameter of 95 nm. The largesurface area of these nanomaterials provides abundant sites for multi-functionality. The surface of nanosensors has beenmodifiedwith positively-charged amino groups in order to facilitate endocytosis and targeting lysosome. Fluorescein is used as theindicator probe for pH measurement, rhodamine B is the probe for temperature, and a europium complex acts as the referencedye. The dual nanosensor responds to pH values in the range from 3.0 to 9.0, and to temperature in the range from 20 to 60 °C.Owing to its good biocompatibility and good sensitivity, the dual nanosensor has been used tomonitor changes in local pH valuesand temperature in the lysosome of HeLa cells.

Keywords Silica nanoparticle . Fluorescein . Rhodamine B . Europium complex . Temperature response . pH response .

Intracellular sensing . Ratiometric measurement . Core-shell

Introduction

Lysosome is a cellular acidic organelle (pH 4.5–5.5) thatis taking care of potential intracellular differentiation, degra-dation of cellular debris, and defends the homeostasis [1].Lysosomes are playing vital rule in recycling of macromole-cules, bone remodeling, intracellular transportation, antigenprocessing, and reparation of plasma membrane [2, 3].Dysfunction of lysosomes has severe impacts on the fate ofcell and links with many diseases [4, 5]. Lysosome pH has

been proved to be one of most important parameters thatmaintain normal activities of lysosome, and variation of pHin lysosome has very close connection with many diseases [6].In addition, it is reported that lysosome pH can be influencedby changing local temperature [7]. Increase of temperaturecauses elevating of lysosomal pH. Therefore, it is importantto compensate temperature influences during pH measure-ment in lysosome. Moreover, researchers have revealed thatthe intracellular distribution of heat is heterogeneous. Thetemperature gap in different regions can reach up to 0.96 °C[8]. Thus, precise imaging of lysosome pH distribution re-quires measuring both pH and temperature accurately, andpreferably, at the same site. With the development ofluminescence-based sensing and imaging technology, opticalnanosensors have become one of the popular approaches forintracellular studies. However, the luminescence properties ofall luminophores are influenced by temperature [9], and thiseffect should be compensated during precise measurement.

The traditional thermocouples are not suitable for measur-ing temperature at micro/nanoscale because of their bulkysize. The development of luminescent nanosized temperaturesensor can fulfil this requirement [9]. However, the use of

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00604-018-3040-y) contains supplementarymaterial, which is available to authorized users.

* Xu-dong Wangwangxudong@fudan.edu.cn

1 Department of Chemistry, Fudan University,200433 Shanghai, People’s Republic of China

2 Key Laboratory of Fine Chemicals, Department of Chemistry, DalianUniversity of Technology, Dalian 116012, People’s Republic ofChina

Microchimica Acta (2018) 185:533 https://doi.org/10.1007/s00604-018-3040-y

temperature nanosensors along with pH nanosensors for intra-cellular studies greatly increases the number of alien materialsinside cells, which will disturb normal cellular activities. Inaddition, two kinds of nanosensors cannot be spatially distrib-uted together. It is difficult to compensate temperature varia-tion during pH measurement. Therefore, integrating pH andtemperature sensor together to form a single dual nanosensorbecomes essential for measuring pH in lysosome.

Construction of the dual nanosensor is much more difficultthan expected, since probes for pH and temperature can havesignificantly different physiochemical properties, and theyshould all chemically match the properties of matrix material.At the same time, the luminescent properties and brightness ofboth probes should be maintained while keeping their size assmall as possible. To date, there are only a few reports onmultiple nanosensors for intracellular studies. The first intra-cellular dual nanosensor was developed by Qian and co-workers [10]. They labelled a pH-sensitive indicator on atemperature-sensitive polymer poly(N-isopropylacrylamide),and produced spherical nanosensors using an emulsion poly-merization technique. The dual sensor can simultaneouslymeasure pH in the range of 5.0–9.0 and temperature from 25to 37 °C. Later on, our group developed the first ultra-smalldual nanosensor for measuring intracellular oxygen and pHdirectly in the cytosol [11]. Owing to the unique sensor design,the nanosensors have a size of only 12 nm, and their surfaceare protected by dense poly(ethyl glycol), which endows thempossessing high stability, good biocompatibility and excellentsensitivity for intracellular study. Following these pioneerwork, intracellular dual nanosensor for pH and temperature[12], for pH and oxygen [13], for glucose and temperature[14], for pH and Cu2+ [15], and for pH and O2

•- [16], haveemerged, and their number is still increasing. However, thesedual nanosensors are mainly fabricated based on polymers,which are chemically inert and difficult to be functionalized.

Herein, we developed a biocompatible and ratiometricdual nanosensor for measuring pH and temperature in lyso-some. The nanosensors are constructed based on easily ac-cessible silica nanoparticle, which are readily available indifferent size and morphology. A reference europium com-plex and a temperature-sensitive probe rhodamine B werecovalently immobilized inside silica nanoparticles. The sur-face was then functionalized with amino groups, which notonly provide reactive groups to covalently link the pH-sensitive probe fluorescein, but also feature the nanosensorswith positively charge, and facilitate cellular uptake andtargeting lysosome. In vitro experiments showed that thenanosensors have good response to pH in the range from3.0 to 9.0, and to temperature in the range from 20 to 60 °C.The constructed nanosensors can be easily taken up byHeLa cells, and located mainly in the lysosomes, whichare then used for monitoring pH and temperature variationinside lysosome. Results have shown that the dual

nanosensors exhibit good ratiometric sensitivity in monitor-ing the two important parameters inside lysosome.

Experimental section

Chemicals and materials

Chemicals in analytical grade and double distilled water (DDW)were used throughout this study. Cyclohexane, 1-octanol, 3-aminopropyltriethoxysilane (APTES), polyethylene glycol, tri-ton X-100, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-drochloride (EDC), N-hydroxysuccinimide (NHS), HCl,europium(III) Chloride hexahydrate (EuCl3·6H2O) andtriethylamine (TEA) were purchased from TCI (www.tcichemicals.com, Shanghai, China). Rhodamine Bisothiocyanate (RBITC) and fluorescein isothiocyanate (FITC)were bought from Heowns (www.heowns.com, Tianjin, China).Tetraethyl orthosilicate (TEOS), N1-(3-trimethoxysilylpropyl)diethylenetriamine (DETAPTMS) were bought from Sigma-Aldrich (www.sigmaaldrich.com, Shanghai, China). N,N,N′,N′-(4′-phenyl-2,2′:6′,2″-terpyridine-6,6″-diyl)bis (methylenenitrilo)tetrakis (acetic acid) (PTTA) were synthesized according to theliterature [17]. For cellular experiments, HeLa (human cervicalcarcinoma cell) cell lines were bought from the cell bank of typeculture collection of the Chinese Academy of Sciences(Shanghai, China). The culture medium (containing minimumessential medium eagle, penicillin-streptomycin, nonessentialamino acid solution and L-glutamine solution), Hanks’ balancedsalt solution (HBSS) and trypsin-EDTA (0.25%) were boughtfrom Sigma-Aldrich. Phosphate buffered saline (PBS) with pHin the range of 7.2–7.4 was bought from BBI Life Sciences(www.bbi-lifesciences.com, Shanghai, China). The CCK-8 cellcounting kit was bought from Dojindo (www.dojindo.cn). Thelysotracker Blue DND 22 was purchased from Thermofisher(www.thermofisher.com).

Instruments

The morphology and size of the nanosensors were character-ized using a Carl Zeiss Ultra 55 field emission scanning elec-tron microscopy (FESEM) (Zeiss, Germany https://www.zeiss.com). Their surface zeta potential was measured usinga Malvern Zetasizer Nano ZS (Malvern Instruments Ltd.,Malvern, UK www.malvern.com/en/). The fluorescencespectrum was recorded on a Hitachi F-7000 FluorescenceSpectrophotometer (Hitachi, Japan https://www.hitachi-hightech.com), and the excitation and emission slit widthwere set at 5 nm. Cell viability was tested using CCK-8 cellviability test kit, and the absorbance was measured on aSynergy H1 Hybrid Multi-Mode Microplate Reader (Biotek,USA https://www.biotek.com). The fluorescence images ofHeLa cells were acquired using a Leica DMi8 fluorescence

533 Page 2 of 9 Microchim Acta (2018) 185:533

microscopy (Leica, Germany ht tps : / /www.le ica-microsystems.com). The filter sets used for microscopicimaging are as follows: FITC (excitation: 450–490 nm,d i c h r o i c : 5 1 0 nm , em i s s i o n : 5 1 0 – 5 4 0 nm ) ,RBITC(excitation: 517–563 nm, dichroic: 580 nm,emission: 565–605 nm), and Eu complex (excitation: 361–389 nm, dichroic: 514 nm, emission: 590–650 nm).

Synthesis of the silane-PTTA-Eu3+ conjugate

The PTTAwas used as a ligand to form conjugate with Eu3+ ionand gave luminescent Eu-PTTA complex. In order to covalentlyimmobilize the Eu-PTTA complex inside silicamatrix, the PTTAligand was further functionalized with silane [18]. Typically,0.8 mg NHS (7.0 μmol) and 4.6 mg EDC (24 μmol) weredissolved in 80 μL ethanol, then 20 μL Na2CO3 buffer(50 mM, pH= 9.5) containing 1.78 mg PTTA (3.0 μmol) wasadded. The mixture was stirred for 1.0 h at room temperature,and then 1.5μLAPTES (6.3μmol) was added. Themixture wasstirring for another 2.0 h. Finally, 100μL of 30mMEuCl3·6H2O(3.0 μmol) aqueous solution was added to form the luminescentconjugate Eu-PTTA-silane.

Reversed micelle method for preparation of dualnanosensor

Firstly, 1.5 μL APTES (6.3 μmol) and 50 μL RBITC solution(10 mM, 0.5 μmol) were mixed in DMSO and kept understirring overnight to produce RB-silane. 1.6 mL of 1-octanol(10 mmol), 1.7 mL triton X-100 (1.8 mmol) and 7.6 mL cyclo-hexane (70 mmol) were mixed in a round flask with stirring,followed by adding 1.5 μL APTES (6.3 μmol) and 300 μLdistilled water. Then, Eu-PTTA-silane conjugate (200 μL) andRB-silane (50 μL) were added and stirred for 30 min. Thesynthesis of luminescent silica nanoparticle is initiated byadding 60 μL NH4OH (28%, 0.80 mmol) and 100 μL TEOS(0.44 mmol), and the solution was stirred for 24 h. The surfacemodification with reactive amino groups was performed by fur-ther adding 50 μLTEOS (0.22 mmol) and 10 μL DETAPTMS(0.039 mmol) into the solution, which was stirred for another24 h. Finally, the obtained Eu-RB@SiO2-NH2 nanoparticleswere harvested via centrifuging and washing steps to removeresidue of surfactants and/or unreacted materials.

The pH-sensitive dye fluorescein was labelled on the sur-face of Eu-RB@SiO2-NH2 nanoparticles by exploring the re-action between isothiocyanate and surface amino groups.Typically, the Eu-RB@SiO2-NH2 nanoparticles were dispersedin dried DMF at a concentration of 10 mg·mL−1, followed byaddition of 0.70 μL of TEA (7.2 μmol) and 2.0 μL FITCsolution (1.0 mM, 2.0 nmol). The mixture was kept in darkand stirred overnight, which was followed by washing thedual nanosensors with DMF for several times and redispersedin ethanol.

In vitro fluorescence measurement

The dual nanosensor were redispersed in 100 mM Britton-Robinson buffer at a concentration of 1.0 mg·mL−1. For pHsensing, the dual nanosensors were excited at 473 nm. As fortemperature measurement, they were excited at 370 nm. Boththe excitation and the emission slit widths on the spectrometerwere set at 5 nm.

Intracellular fluorescence imaging

The HeLa cells were cultivated in the standard culture media(87% minimum essential medium eagle, 10% FBS, 1% L-glutamine solution, 1% penicillin-streptomycin solution and1%MEMnonessential amino acid solution) for 24 h. Then theculture media was changed into standard culture media con-taining 200 μg mL−1 dual nanosensors, and cells were incu-bated for 4 h. After that, the cells were washed carefully withthe HBSS for three times to remove free nanosensorssuspended in the media and were ready for fluorescenceimaging.

For cell viability test, HeLa cells were incubated with thedual nanosensors at varies concentrations (0, 50, 100, 200 and300 μg mL−1) for 24 h, and the experiment is repeated forthree times. The cell viabilities were tested by adding CCK-8cell viability test kit and incubated for another 2 h. The absor-bance at 450 and 650 nm were recorded, and absorbance at650 nm is used as reference for background subtraction.

For the colocalization study, the nanosensors are incu-bated with HeLa cells at a concentration of 200 μg mL−1

at 37 °C for 4 h, then the HeLa cells with uptakennanosensors were further stained with lysotracker BlueDND 22 at a concentration of 50 nM for 30 min. Freedyes were washed away using PBS, then the cells wereimaged using the fluorescence microscope.

For intracellular pH response study, HeLa cells were treat-ed with the potassium-rich solutions at different pH values for30 min. The solution contains 10 μg mL−1 nigericin was usedto manipulate intracellular pH value. The potassium-rich so-lution composes of 130 mM potassium chloride, 10 mM so-dium chloride, 1 mM magnesium sulfate and 10 mM Na-MOPs [19]. For intracellular temperature response study, thetemperature of the object stage was controlled at 4 and 40 °C,respectively, using a circulating system with precise tempera-ture control.

Result and discussion

Choice of materials

The scheme of designing the targeted dual nanosensor isshown in Fig. 1a. Silica nanoparticles are selected as

Microchim Acta (2018) 185:533 Page 3 of 9 533

supporting matrix for nanosensors because of their good bio-compatibility, optical transparence, excellent size and mor-phology control. The surface of silica nanoparticles can beeasily functionalized using the well-established silane tech-nique. The europium complex was selected as reference dyesbecause of its extremely sharp emission at around 612 nm. Itslong lifetime enables intracellular nanosensor tracking andlocalization based on time-gated technology [18], and strongautofluorescence from cells can be eliminated. Rhodamine Bwas selected as the temperature probe, because it is one of themost sensitive temperature organic probes in physiologicaltemperature range. This probe can be excited using visuallight, and exhibits high quantum yield and good brightness[9]. The probe is insensitive to pH in the physiological range[20] (see Fig. S1 in the supporting information), and incorpo-ration of the probe inside silica nanoparticle can furtherprotected them from interacting with media. At the end, fluo-rescein is chosen as the pH probe because of its good bright-ness, easy accessibility, high quantum yield and suitable pKafor intracellular studies. [21, 22] In order to prevent signalcross-talking and the influences of ions, proteins, and otherintracellular molecules on the luminescence of probes, thetemperature probe Rhodamine B and the reference dyeEuropium complex are covalently immobilized inside silicananoparticles, which not only prevents dye from leaching andaggregation, but also protects them from potential quenchersand FRETeffect between dyes [23]. The pH probe fluoresceinis labelled on the surface of nanosensors, and directly exposed

to protons, which is beneficial to proton diffusion and pHmeasurement.

The characterization of dual nanosensor

The preparation of the nanosensor is straightforward. RhodamineB and the europium complex are covalently linked with APTES,and then immobilized inside silica nanoparticles via a one-potreaction. Afterwards, the surface of the nanoparticles is function-alizedwith amino groups and chemically linkedwith fluorescein.Figure 1b shows the scanning electron microscopy image of theprepared dual nanosensors. They showed spherical shape anduniform size with average size of 91 ± 6 nm (calculated from100 particles). More importantly, the aqueous suspension of thedual nanosensors is stable for at least one month without ob-served precipitation or aggregation, as indicated from dynamiclight scattering measurement in Fig. 1c. It is noted that thenanosensor size characterized using DLS is larger than that usingelectron microscopy, because the hydrodiameter of particles ismeasured in DLS measurement.

The surfacemodification of amino groups gave the nanosensora net positive surface charge, as indicated by surface zeta potentialmeasurement (29.1 mV, Fig. S2 in the supporting information).The positively-charged surface of the dual nanosensors facilitatestheir interaction with negatively-charged cell membranes, and isbeneficial for nanosensor uptake. In addition, the reactive aminogroups can be used for immobilizing pH-sensitive probe. Part ofthe surface amino groups are labelled with fluorescein to form the

RBITC

Eu-PTTA-APTES

FITCSiO2

a b

c

Fig. 1 The scheme (a) and field-emission scanning electron mi-croscopic image (b) and size dis-tribution measured via dynamiclight scattering (c) of the dualnanosensors

533 Page 4 of 9 Microchim Acta (2018) 185:533

dual nanosensor and the rest are useful to promote cellular uptake.Since all fluorescein molecules are located on the surface ofnanosensors, they can easily become protonated/deprotonated,and sense fluctuation of protons in local environment.

Ratiometric luminescence responses to pHand temperature

We then studied the response of the dual nanosensor to pH andtemperature. The luminescence spectra of dual nanosensor isshown in Fig. 2a. Once excited at 370 nm, there are threeemission peaks at wavelength of 512 nm, 588 nm and612 nm, corresponding to the luminescence of fluorescein,Rhodamine B and Eu-PTTA, respectively. The two shoulderpeaks at 588 nm and 595 nm belong to the emission of Eu-PTTA complex. The responses of the dual nanosensor towardstemperature change were studied in detail on a fluorescence

spectrometer. As shown in Fig. S3 in the supporting informa-tion, the luminescence intensities of all three dyes are decreas-ing when temperature are elevated from 20 to 60 °C, since thenon-radiative relaxation rate of all luminescent dyes is in-creased with rising temperature. The luminescent intensitiesis normalized at 612 nm, and the result is shown in Fig. 2a. Itis obvious that the RhB dye exhibits better temperature sensi-tivity, with intensity change of 1.2% per degree at 37 °C. Therelationship between fluorescence ratio (I588 nm/ I612 nm) andtemperature fits well with the Arrhenius equation. Theratiometric measurement of temperature can significantly re-duce the influences of light source fluctuation, optical align-ment, autofluorescence, as well as probe uneven distribution.

Because of relative narrow excitation spectra, fluoresceincannot be excited efficiently at 370 nm, and the nanosensorshows small signal change at different pH once excited at thiswavelength (see Fig. S4 in the supporting information). This

Fig. 2 The fluorescence spectra of the dual nanosensor at different temperature (a, excited at 370 nm), and at different pH (c, excited at 473 nm),respectively. The corresponding ratiometric calibration curve for temperature (b) and pH (d) are measured based on 3 independent measurements

Microchim Acta (2018) 185:533 Page 5 of 9 533

makes ratiometric measurement of both pH and temperatureusing a single wavelength excitation difficult. In order to en-hance pH sensitivity, the dual nanosensors are excited at473 nm. As shown in Fig. 2c, the nanosensors emitted strong-ly at 512 nm and 588 nm, and the green emission increasedsignificantly when pH increasing. In contrast, the emission ofRhB almost has no change, which acts as reference for pHsensing. The calibration curve plotted from fluorescence ratio(I512 nm/ I588 nm) and pH shows the typical Henderson-Hasselbalch type response. The pKa value of the nanosensorsis 6.65, which is slightly higher than that of free fluorescein inaqueous solution (6.5), and is mainly attributed to theremaining amino groups on particle surface. It is knownthat the pH value in lysosome usually around 4.5, and thenanosensor is still sensitive in this acidic condition withintensity ratio increased from 0.90 to 1.45 when the pHchanges from 4 to 5.5. In addition, the measurement erroris smaller in acidic environment, which is beneficial foraccurate measurement.

Figure S5 in the supporting information showed thephotostability of the nanosensors, all the dyes showed goodphotostability, including fluorescein, which is mainly due tothe formation of nanoparticles. Compared with fluoresceindyes in solution, the fluorescein modified on the silica nano-particles has better photostability with only 7% decrease afterone hour of continues exposure to incident light at 473 nm.The good photostability of the nanosensor ensures that thenanosensor can be used for continuously measuring these pa-rameters for long-term studies. To further study the selectivityof dual nanosensors, influences of oxygen and protein adsorp-tion on sensor performance were studied in details. As shownin Fig. S6 and S7, the presence of oxygen and protein do notobviously influence pH and temperature response of thenanosensor.

Cytotoxicity and intracellular localization of dualnanosensor

Before applying the dual nanosensors for intracellularstudies, they were firstly investigated for their cytotoxic-ity and results are summarized Fig. S8 in the supportinginformation. HeLa cells maintain more than 95% of theirinitial viability even when the nanosensor concentrationreaches up to 0.3 mg mL−1, demonstrating that the dualnanosensors have low cytotoxicity and good biocompati-bility. We further studied the distribution of the dualnanosensors inside HeLa cells. After uptake, the cellsare co-stained with the commercial-available lysotrackerblue dye. Result (Fig. S9 in the supporting information)showed that the RhB emission overlaps well with that oflysotracker blue, and the Spearman’s rank correlation co-efficient is 0.76, indicating that the dual nanosensors aremainly distributed in lysosomes.

Fluorescence ratiometric imaging of nano-sensorstowards pH and temperature in HeLa cells

Because of their low cytotoxicity, efficient cellular uptake andgood brightness, the dual nanosensors are finally subjected tomeasure pH and temperature variations inside cells. The intra-cellular pHwas adjusted using nigericin [24, 25]. As shown inFig. 3, the green fluorescence of fluorescein enhanced signif-icantly with pH increased from 4.0 to 9.0. In contrast, theluminescence brightness of RhB and Eu-PTTA almost re-mains the same. The calculated image ratio between FITCchannel and RhB channel showed the pH changemore clearly,which proved that the nanosensor is able to ratiometricallymeasure intracellular pH.

The response of the dual nanosensors to temperature wasperformed in a similar way. The temperature of cell culturemedia is precisely controlled using a water-circulating bath,and HeLa cells are imaged on the microscope at differenttemperatures. As shown in Fig. 4, the brightness of RhB de-creased obviously with rising temperature, while fluoresceinand Eu-PTTA showed slight intensity change. The ratio imagecalculated from RhB channel and Eu channel showed obvi-ously temperature differences. The fake red spot shown at40 °C in the ratio image is mainly due to saturation of theCCD camera. In addition, from these intracellular images, itis obvious that the fluorescence from all three dyes overlappedvery well with each other, indicating that these dyes are cova-lently immobilized on the same particle. This endows thatnanosensor can measure intracellular pH and temperature si-multaneously and, more importantly, at the same site. In com-bination with their high biocompatibility, good brightness,good photostability and high sensitivity, the dual nanosensorare useful tools for lysosomal pH and temperature sensing andimaging, and are suitable for long-term monitoring of thesetwo parameters.

At the end, we compared the-state-of-the-art dualnanosensor for pH and temperature sensing. As summarizedin Table 1, it is obvious that not so many dual nanosensorshave been developed so far for simultaneously measuring in-tracellular pH and temperature. These nanosensors either suf-fer from difficulties in preparation (including polymerizationand labelling) and application, or from severe signal cross-talking, which are difficult to use for intracellular studies. Incontrast, our nanosensors can be easily prepared and the sur-face functionalization technology is already well established.The remaining surface functional groups on nanosensors canbe further explored for cellular active-targeting, carryingdrugs, imaging, and tracking purposes. The major limitationof the nanosensors is the short wavelength excitation for theeuropium complex, which can be solved with the rapid devel-opment of synthetic dyes, such as megastokes dyes [26], andprovide the opportunity to excite all three dyes at a singlewavelength, or even using a laser line.

533 Page 6 of 9 Microchim Acta (2018) 185:533

Conclusions

In summary, we have developed a novel targeted dualnanosensor for imaging intracellular pH and temperature si-multaneously. The nanosensor was constructed using silicananoparticle as supporting material. Both the referenceEuropium complex and the temperature-sensitive probeRhodamine B are covalently immobilized inside silica nano-particles, which shields them from interacting with intracellu-lar molecules, and prevents potential quenching and solventseffects. The long luminescent lifetime of the europium com-plex provides additional feature for nanosensor tracking and

localization based on time-gated technique, which significant-ly reduces autofluorescence background. The nanosensor sur-face was modified with positively-charged amino groups, andpart of that were labelled with pH-sensitive fluorescein. Sincefluorescein molecules directly exposed outside, thennanosensors showed good sensitivity to pH change. Owingto their low cytotoxicity, good biocompatibility, high sensitiv-ity and good photostability, the nanosensors have been suc-cessfully used for ratiometrically measuring pH and tempera-ture in lysosome. Themeasured temperature value can be usedto calibrate pH response. Because of their ease of preparationand high performance, these nanosensors are useful tools to

25 C

37 C

50µm

Fluorescein Rhodamine B Eu Ratio image

40 C

25 C

40 C Fig. 4 The temperature response of dual nanosensors in HeLa cells. The ratio images are calculated by dividing the Rhodamine B channel to the Eucomplex channel, and represented in presudo color

pH 4

Fluorescein Rhodamine B Eu Ratio image

pH 7

pH 9

50 m pH 4

pH 9

Fig. 3 The pH response of dual nanosensors in HeLa cells, the intracellular pH was adjusted with Nigericin. The ratio image was calculated from theFluorescein channel and RhB channel using ImageJ software

Microchim Acta (2018) 185:533 Page 7 of 9 533

Table1

Anoverview

ofnanomaterial-basedmethods

forpH

andtemperature

imaging

Materials

Method

MeasurementR

ange

Merits

Lim

itatio

nsRef.

Pseudorotaxane-likearchitecturefabricated

from

a1,5-diam

inonaphthalene

end-functio

nalized

poly(N

-isopropyl)acrylam

ideand

cyclobis(paraquat-p-phenylene)

Absorption/Visiblecolor

change

Temperature

(color

change

whenT>Tcloudpoint),

pHvalues

(1.0–7.0)

Directv

isiblereadout

Donotsensing

temperature

continuously;

unknow

npH

sensitivity,bulky

polymer

notsuitableforintracellularapplications

[27]

2-{5-[4-((4-

-nitrophenyl)diazenyl)phenyl]-1,3,4--

oxadiazol-2-ylthio}ethyl

acrylate

copolymerized

with

N-isopropylacrylamide

Absorption/Fluorescenceim

-aging

Temperature

(40–75

°Cat

pH4–12;3

0–65

°Cat

pH2),pHvalues

(2.0–10.0)

Water-soluble,visualcolor

change

Difficulties

infunctio

nalizationand

preparation,poor

temperature

response,

bulkypolymer

notsuitablefor

intracellularapplications

[28]

Poly(N-isopropylacrylamide)

/poly(acrylic

ac-

id)nanogels

Hydrodynamicdiam

eters

measurement

Temperature

(20–50

°C),pH

values

(3.0–7.0)

pHandTemperature-triggered

volumephasetransitio

n;Less

mutualinterference

Not

possibleto

measure

particlesize

invivo

[29]

Disperseredpolymerized

onPo

ly(olig

oethyleneglycolm

ethacrylate)

Absorption/Color

change

Temperature

(10–20

°C)pH

values

(1.0–7.0)

Visualcolor

change,good

stability

Solvatochrom

iccolorchange

will

lost

whenhydrophilicity

ofthecopolymer

increased;

Not

suitableforintracellular

application

[30]

Film

basedsensor

incorporated

with

Ru(dpp)-doped

polyacrylonitrile

Nanoparticles,surfacefunctio

nalized

with

fluoresceinforpH

sensing

Lifetim

emeasurement

Temperature

(5–55°C

),pH

values

(4.0–9.0)

Goodsensitivity,highselectivity

andno

signalcross-talking

Difficultin

particlepreparationandsurface

functio

nality,poor

size

control,not

suitableforintracellularstudies.

[12]

SeminaphthorhodafluoraspH

indicator,Cr(III)-

activ

ated

gadolin

ium

alum

inium

borateas

temperature

probeandreferencedye

DualL

ifetim

eReferencing

Temperature

(5–45°C

),pH

values

(4.0–9.0)

Red

light

excitatio

n;Turnable

dynamicrange;Chemicaland

photochemicalinertness

P oor

photostability,film

basedsensor,not

suitableforintracellularstudies.

[31]

Carbondots

Fluorescenceim

aging

Temperature

(10–82

°C),pH

values

(low

erthan

6.0or

higher

than

8.6)

Asingletcarbondotscanbe

used

forpH

,tem

perature

andiron

ionsensing

Not

possibleto

measure

pHand

temperature

atthesametim

e,severe

signalcross-talking,shortexcitatio

nwavelengths,poorstabilityandquantum

yield.

[32]

Europium

complex

andRhodamineB

covalently

immobilizedinside

silica

nanoparticles.Nanosensorsurfacelabelled

with

fluorescein

Ratiometricfluorescence

imaging

Temperature

(20–60

°C),pH

values

(3.0–9.0)

Highsensitivity,goodselectivity,

high

stability

andno

leaching

ofdyes,precise

surface

chem

istryandsize

control,

ease

offunctio

nalization

Shortw

avelengthexcitatio

nof

reference

dye,butcan

beim

proved

usingnew

synthetic

dyes.

thiswork

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study intracellular parameters, which would be helpful to bet-ter understanding the fundamental mechanism of lysosomalfunctionalities.

Acknowledgements This work was financially supported by NationalKey R&D Program of China (2017YFC0906800), National NaturalScience Foundation of China (21775029), the Recruitment Program ofGlobal Experts (1000 Talent program) in China, and the Program forProfessor of Special Appointment (Eastern Scholar) at ShanghaiInstitutions of Higher Learning (No. TP2014004), which are greatlyacknowledged.

Compliance with ethical standards The author(s) declare thatthey have no competing interests.

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