Multiplexed and amplified chemiluminescence resonance energy transfer (CRET) detection of genes and microRNAs using dye-loaded hemin/G-quadruplex-modified UiO-66 metal–organic framework nanoparticles†

Dye-loaded UiO-66 metal–organic framework nanoparticles (NMOFs) modified with catalytic hemin/G-quadruplex DNAzyme labels act as functional hybrid modules for the chemiluminescence resonance energy transfer (CRET) analysis of miRNAs (miRNA-155 or miRNA-21) or genes (p53 or BRCA1). The dye-loaded NMOFs (dye = fluorescein (Fl) or rhodamine 6G (Rh 6G)) are modified with hairpin probes that are engineered to include in their loop domains recognition sequences for the miRNAs or genes, and in their stem regions caged G-quadruplex domains. In the presence of the analytes miRNAs or genes, the hairpin structures are opened, leading, in the presence of hemin, to the self-assembly of hemin/G-quadruplex DNAzyme labels linked to the dye-loaded NMOFs. In the presence of luminol and H2O2, the hemin/G-quadruplex DNAzyme labels catalyze the generation of chemiluminescence that provides radiative energy to stimulate the process of CRET to the dye loaded in the NMOFs, resulting in the luminescence of the loaded dye without external excitation. The resulting CRET signals relate to the concentrations of the miRNAs or the genes and allow the sensitive analysis of miRNAs and genes. In addition, the DNA hairpin-functionalized dye-loaded NMOF sensing modules were further applied to develop amplified miRNA or gene CRET-based sensing platforms. The dye-loaded NMOFs were modified with hairpin probes that include in their loop domain the recognition sequences for miRNA-155 or miRNA-21 or the recognition sequences for the p53 or BRCA1 genes. Subjecting the hairpin-modified NMOFs to the respective miRNAs or genes, in the presence of two hairpins Hi and Hj that include in their stem regions caged G-quadruplex subunit domains, results in the analyte-triggered opening of the probe hairpin linked to the NMOFs, and the opened hairpin tethers induce the cross-opening of the hairpins Hi and Hj by the hybridization chain reaction, HCR, resulting in the assembly of G-quadruplex wires tethered to the NMOFs. The binding of hemin to the HCR-generated chains yields hemin/G-quadruplex DNAzyme wires that enhance, in the presence of luminol/H2O2, the CRET processes in the hybrid nanostructures. These amplification platforms lead to the amplified sensing of miRNAs and genes. By mixing the Fl- and Rh 6G-loaded hairpin-functionalized UiO NMOFs, the multiplexed CRET detection of miRNA-155, miRNA-21 and the p53 and BRCA1 genes is demonstrated.


Introduction
Substantial research efforts have been directed in the past two decades towards the development of sensors for the detection of DNA, RNA, microRNA (miRNA) and aptamer-based sensors. 1 Numerous electrochemical, 2 optical, 3 microgravimetric 4 and magnetic eld-based sensors 5 were developed, and ingenious amplication methods were demonstrated for the ultrasensitive detection of DNA-based sensors. 6 Among the optical DNA-based sensors, uorescence-based sensing platforms using FRET mechanisms, 7 the application of luminescent quantum dots 8 and luminescent Ag nanoclusters, 9 and the use of DNAzymecatalyzed chemiluminescent 10 sensing platforms were demonstrated. In addition, optical plasmon-based DNA sensors involving absorbance changes associated with the aggregation of plasmonic particles and plasmonic coupling between nanoparticles 11 and surface plasmon resonance transduction 12 led to highly sensitive DNA sensing systems. Amplied DNA sensing schemes have included the application of DNA machineries, such as the replication/nicking machinery, 13 rolling circle amplication, 14 autocatalytic hybridization chain reaction (HCR), 15 the biocatalytic regeneration of nucleic acid analytes in the presence of exonuclease, 16 and so on. In addition, the development of multiplexed analysis schemes for the detection of genes or miRNAs has attracted continuous efforts. Different sized semiconductor quantum dots 17 or silver nanoclusters, 18 exhibiting size-controlled luminescence functions, and the selective desorption of uorophores from graphene oxide supports 19 were used to design multiplexed sensing assays. Also, different DNAzymes associated with DNA machineries or DNA nanostructures were used for multiplexed analysis of genes. 20 In addition, the chemiluminescence resonance energy transfer (CRET) process was reported to be a useful path for the multiplexed analysis of genes. 21 The hemin/G-quadruplex-modied semiconductor quantum dot hybrids act as useful functional structures for activation of the luminescence of the quantum dots through the chemiluminescence energy transfer path. The hemin/G-quadruplex-stimulated generation of chemiluminescence, upon the catalyzed oxidation of luminol by H 2 O 2 , provides a useful energy source for the excitation of the quantum dots with direct excitation. Accordingly, by the modication of different-sized semiconductor quantum dots functionalized with hairpin DNA probes that include target-specic sensing domains and caged G-quadruplex sequences, the multiplexed CRET analysis of different genes was demonstrated. 21 The detection of miRNAs is of particular interest. miRNAs are short noncoding RNA sequences (19-26 bases) that control gene expression in various cellular transformations. 22 The upregulation or down-regulation of miRNAs has been related to biological processes, such as proliferation or apoptosis and various diseases. In particular, sequence-specic miRNAs are biomarkers for various types of cancer cell. Indeed, substantial research efforts have been directed to the development of sensing platforms for miRNAs. 23 The low level of miRNAs in cancer cells requires, however, the development of analytical amplication methods and different pathways to detect miR-NAs by exponential amplication using endonuclease, 24 the application of the hybridization chain reaction (HCR) 25 and catalytic hairpin assembly were demonstrated. 26 Recent reports applied photoactivated toehold-mediated strand displacement and DNAzyme-driven nanomotors as amplication methods for analyzing miRNAs. 27 Metal-organic framework nanoparticles, NMOFs, represent a class of porous materials 28 that nd broad applications in catalysis, 29 storage and separation of gases, 30 drug delivery carriers 31 and sensing. 32 Recently, the synthesis of Cu 2+ -bipyridine NMOFs, exhibiting horseradish peroxidase activities re-ected by the generation of chemiluminescence upon the catalytic oxidation of luminol by H 2 O 2 , was reported. 33 Upon the postmodication of bipyridine ligands with Cu 2+ ions, the Cu 2+ -modied NMOFs yielded a functional module for inducing the CRET in the hybrid carriers and the generation of the uorescein uorescence without external excitation. Nonetheless, no sensing functions by these hybrid carriers were demonstrated. Modied NMOFs with signal-triggered recongurable nucleic acids have recently been introduced as stimuli-responsive drug carriers. 34 Different triggers to unlock nucleic acid-gated NMOFs were discussed, including the pH-induced dissociation of i-motif or triplex nucleic acid gates, 35 and the use of miRNAs 36 or ligand-aptamer complexes 37 to unlock nucleic acid-gated NMOFs were reported.
In the present study, we introduce dye-loaded UiO-66 NMOFs 38 modied with hemin/G-quadruplex units as hybrid modules that act as functional nanostructures for the generation of CRET. We demonstrate the ON/OFF switchable generation of CRET by the hybrid nanoparticles. In addition, through the engineering of functional nucleic acid hairpin structures on the NMOFs, we introduce CRET-based miRNA and gene sensing platforms. By designing mixtures of appropriately engineered nucleic acid hairpin-modied dye-loaded NMOF hybrids, the multiplexed CRET analysis of miRNAs and genes is demonstrated. Furthermore, by coupling the HCR to the hairpin-modied dye-locked NMOF modules, the amplied CRETbased sensing of miRNAs or genes is achieved. The novelty of the study rests on the unique properties of hemin/G-quadruplex to catalyze the generation of chemiluminescence and the feasibility to conne, in the porous structure of UiO-66 NMOFs, a high loading of dyes in proximity to the hemin/G-quadruplex modiers, a hybrid composite that allows effective CRET and multiplexed detection of the analytes. It should be noted that recently, hemin/G-quadruplex modied NMOFs were applied for the amplied chemiluminescent imaging of miRNA-133a in sera. 39 In contrast to this study, our amplied hemin/Gquadruplex NMOF hybrid miRNA and gene sensing platforms apply the CRET mechanism as a readout signal, which allows the multiplexed analysis of different miRNAs or genes via the encapsulation of different CRET acceptor dyes in the NMOF carriers.  terephthalic acid. The 5 0 -end of the nucleic acid (1) was phosphorylated and was ligated to the vacant Zr 4+ -ion ligation sites on the NMOFs. Bipyramidal UiO-66 NMOFs were formed; Fig. 1(B) and dynamic light scattering experiments (Fig. S1 †) indicate an average diameter of 200 nm for the nanoparticles. The crystallinity of the UiO-66 NMOFs was determined by powder X-ray diffraction (XRD, Fig. 1(C)). The zeta-potential of the NMOFs before and aer modifying with (1) corresponded to À5 mV and À30 mV, respectively ( Fig. 1(D)).

Results and discussion, and experimental
The strand (1) is guanosine-rich. In the presence of K + -ions, the strands assemble into G-quadruplex units that associate with hemin and form hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme units. 40 The DNAzyme units catalyze the oxidation of Amplex Red (2) by H 2 O 2 to the uorescent resorun, (3) (Fig. 2(A)). Treatment of the hemin/Gquadruplex-modied NMOFs with 18-crown-6-ether (CE) that eliminates the K + -ions from the G-quadruplex via complexation with the CE 41 results in the separation of the G-quadruplex, leading to catalytically inactive NMOFs. Fig. 2(B) curve (i) shows the K + -ions-stimulated formation of the active hemin/Gquadruplex DNAzyme structure that catalyzes the oxidation of Amplex Red to resorun. Curve (ii) shows the catalytically inactive conguration of the (1)-functionalized NMOFs, and curve (iii) shows the reverse deactivation of the DNAzyme upon CE separation of the G-quadruplex. The switchable activation and deactivation of the hemin/G-quadruplex peroxidase activities could be switched to "ON" and "OFF" states for three cycles, in the presence of K + -ions/CE with no noticeable effect on the catalytic activity of the DNAzyme. It should be noted that the hemin/G-quadruplex-catalyzed oxidation of Amplex Red by H 2 O 2 to the uorescent resorun was used to evaluate the loading of (1) on the UiO-66 NMOFs. The (1)-modied UiO-66 NMOFs were, then, loaded with uorescein (Fl) or rhodamine 6G (Rh 6G). Since the unfolded (1) strand includes the guanosine (G)-rich sequence, the addition of K + -ions to the Fl-or Rh 6G-loaded NMOFs resulted in the selfassembly of K + -ions-stabilized G-quadruplex structures that acted as gates to lock the dyes in the NMOFs. Aer extensive washing of the NMOFs, the loading of the NMOFs with Fl and Rh 6G was evaluated to be 80 nmol mg À1 and 70 nmol mg À1 NMOFs, respectively (see Fig. S3 and S4 † and accompanying discussion). The hemin was then incorporated into the Gquadruplex associated with the NMOFs. As shown in Fig. 3(A) and (C), the resulting hemin/G-quadruplex units catalyzed the generation of chemiluminescence through the catalyzed oxidation of luminol (l em ¼ 420 nm), and the resulting chemiluminescence stimulated the chemiluminescence resonance energy transfer (CRET) to Fl or Rh 6G reected by the CRETstimulated emission of Fl (l em ¼ 520 nm) or Rh 6G (l em ¼ 550 nm) ( Fig. 3(B) and (D)), respectively. It should be noted that dye-loaded NMOFs modied with single-stranded nucleic acids do not provide effectively locked porous structures, and the dye undergoes release (leakage) within several hours. Nonetheless, duplex, duplex-based hairpin or G-quadruplex gated NMOFs provide effective locks that prevent the leakage of the dye, due to steric closure of the pores. It should be noted that the dyes are locked in the pores separated by the octahedral subunits comprising the UiO-66 NMOFs and locked in the pores by the DNA locks, rather than being trapped in the octahedral subunits of the particles. Nonetheless, for the CRET process, the dyes have to be conned in the void volumes bridging the octahedral subunits of the NMOFs and for schematic presentation to preserve the dyes in conned nanoenvironments, we introduce them schematically into the octahedral subunits.
The successful hemin/G-quadruplex CRET-stimulated generation of uorescence of the dyes encapsulated in the  The spectrum of chemiluminescence generated by the hemin/G-quadruplex-catalyzed oxidation of luminol, l em ¼ 420 nm, and the CRET luminescence signal of Fl at l em ¼ 520 nm. (C) Schematic CRET between the chemiluminescence generated by the hemin/G-quadruplex-catalyzed oxidation of luminol by H 2 O 2 and the Rh 6G-loaded UiO-66 NMOFs. (D) The spectrum of chemiluminescence generated by the hemin/Gquadruplex-catalyzed oxidation of luminol, l em ¼ 420 nm, and the CRET luminescence signal of Rh 6G at l em ¼ 550 nm.
NMOFs was then used to develop CRET-based microRNA (miRNA) and gene sensing systems, and multiplexed sensing platforms. Specically, we focused on the detection of miRNA-155, overexpressed in HepG2 liver cancer cells, and miRNA-21, overexpressed in MDA-MB-231 breast cancer cells. Fig. 4(A) shows the CRET-based sensing platform for miRNA-155. The UiO-66 NMOFs were functionalized with the phosphorylated tether (4) and loaded with Fl. The Fl-loaded (4)-modied NMOFs were hybridized with hairpin hp1 to lock the dye in the NMOFs. No leakage of the dye from the (4)/hp1-gated Fl-loaded NMOFs could be detected (see Fig. S5 †). Hairpin hp1 was engineered to include in its loop domain the sequence recognizing miRNA-155. The G-quadruplex sequence was embedded, and caged, in the stem region of hairpin hp1. The caged structure of the Gquadruplex sequence prohibits the formation of the Gquadruplex module. In the presence of miRNA-155, the hp1 structure is opened, resulting in the uncaging of the stem domain, and in the presence of K + -ions and hemin, the hemin/ G-quadruplex unit is formed on the surface of NMOFs. This leads to the hemin/G-quadruplex catalyzed oxidation of luminol by H 2 O 2 and to the formation of chemiluminescence that stimulates CRET process to the embedded dye and the uorescence of Fl (l em ¼ 520 nm). Fig. 4(B) shows the chemiluminescence and CRET signals generated by the Flloaded UiO-66 NMOFs upon sensing different concentrations of miRNA-155. As the concentration of miRNA increases, the CRET signal is higher, consistent with the enhanced uncaging of the NMOFs and the increased content of the CRETgenerating catalyst, the hemin/G-quadruplex. Fig. 4(C) shows the derived calibration curve. The system allowed the sensing of miRNA-155 with a detection limit that corresponds to 1.7 nM. (Detection limits in the study followed IUPAC guidelines; 42 see the ESI. †) The sensing of miRNA-155 by the Fl-loaded NMOFs was selective. Treatment of the NMOFs with a foreign miRNA, such as miRNA-145, did not lead to any CRET signals (see Fig. S6 †).
Using the same procedure, the (4)-modied NMOFs were loaded with Rh 6G, and the Rh 6G-loaded NMOFs were locked with a second hairpin (hp2), designed to detect miRNA-21 (for the schematic conguration of the miRNA-21-responsive NMOFs see Fig. S7, ESI †). Hairpin hp2 includes, in its loop region, the miRNA-21 recognition sequence, and in its stem region, the caged G-quadruplex inactive sequence. miRNA-21 is overexpressed in MDA-MB-231 breast cancer cells, and, thus, provides a biomarker for the detection of these cells. Thus, the Rh 6G-loaded (4)/hp2-gated NMOFs were applied to sense miRNA-21. The chemiluminescence spectra and CRET spectra generated by the DNAzyme-modied NMOFs are presented in Fig. 4(D) and the derived calibration curve is shown in Fig. 4(E). The NMOFs enabled the analysis of miRNA-21 with a detection limit that corresponded to 6.7 nM.
The low concentrations of miRNAs in biological samples require, however, the development of amplication paths for the analysis of miRNAs. Different methods to amplify miRNA detection platforms were reported, and these included the regeneration of the miRNAs using exonuclease III, Exo III, and a rolling circle amplication process. As the CRET signal is controlled by the concentration of the hemin/G-quadruplex catalyst units, we argued that the increase of the hemin/Gquadruplex generated by a single opening event of the hairpin probes could enhance the CRET signal, and thereby, improve the sensitivity of the sensing platform. Accordingly, we applied the hybridization chain reaction (HCR) as a path to amplify the miRNA detection platform (Fig. 5(A)). The hairpin, hp 5, was linked to the Fl-loaded anchor (5)-modied UiO-66 NMOFs. The loop of hp5 was engineered to include the miRNA-155 recognition sequence. In the presence of miRNA-155, the hairpin is opened, leading to a free tether that triggers, in the presence of hairpins hp3 and hp4, the HCR process, where the crossopening of hp3 and hp4 leads to the oligomers of selfassembled G-quadruplex chains that catalyze the generation of chemiluminescence, and to the subsequent process of CRET to the Fl dye-loaded NMOFs carriers, leading to the uorescence of Fl (l em ¼ 520 nm). The HCR-stimulated formation of multiple hemin/G-quadruplex catalytic units amplies the resulting CRET signal. Fig. 5(B) shows the chemiluminescence and CRETinduced Fl uorescence spectra generated by the NMOFs at different concentrations of miRNA-155. The resulting calibration curve is shown in Fig. 5(C) (curve (a)). For comparison, in Fig. 5(C), curve (b) shows the calibration curve corresponding to the CRET analysis of miRNA-155 by the single hemin/Gquadruplex-labeled Fl-loaded NMOFs, e.g. Fig. 4 (similar content of Fl-loaded NMOFs). Evidently, the CRET signals for sensing miRNA-155 are signicantly amplied by the HCR amplication path. The HCR-amplied generation of the Cr CRET signal allowed the analysis of miRNA-155 with a detection limit corresponding to 0.17 nM. Thus, the application of the HCR amplication path allowed a 10-fold improvement in the detection limit of miRNA-155.
Using the same approach, the Rh 6G-loaded NMOFs were modied with hairpin hp6, which was engineered to amplify the sensing of miRNA-21, through the HCR process, and the accompanying CRET signal (uorescence of Rh 6G at l ¼ 550 nm) (Fig. S8 †). The resulting chemiluminescence and Rh 6G chemiluminescence resonance uorescence spectra at different concentrations of miRNA-21 and the resulting calibration curve are shown in Fig. 5(E) curve (a). The system allowed the analysis of miRNA-21 with a detection limit of 1.67 nM, a sensitivity value that is ca. 4-fold higher as compared to the detection limit demonstrated by the single hemin/G-quadruplex functionalized NMOFs (Fig. 5(E), curve (b)).
The HCR amplied sensing of miRNA-155 or miRNA-21 was then applied for the multiplexed analysis of the two miRNAs (Fig. 6). A mixture of Fl-loaded miRNA-155-responsive NMOFs and Rh 6G-loaded miRNA-21-responsive NMOFs was exposed to miRNA-155 and/or miRNA-21. In the presence of miRNA-155, only the HCR process associated with the hp5-functionalized Fl-loaded NMOFs is activated, leading to the CRET signal of Fl ( Fig. 6(A), panel I and panel II). In the presence of miRNA-21, only the HCR process associated with the hp6-modied Rh 6G-loaded NMOFs is activated, generating the CRET-induced signal of Rh 6G (Fig. 6(B), panel I and panel II). In the presence of miRNA-155 and miRNA-21, the HCR process associated with the two kinds of NMOF is activated, leading to the CRETstimulated uorescence of Fl and Rh 6G ( Fig. 6(C), panel I and panel II). Note that due to the different CRET signal intensities of the uorophores, the mixture of Fl/Rh 6G-loaded NMOFs consisted of a ratio of 1 : 3 to obtain a clear separation of the CRET signals generated by the two uorophores. (For the inefficient multiplexed analysis of miRNA-155 and miRNA-21 by  the non-amplied mixture of NMOFs described in Fig. 4 and S7, see ESI Fig. S9 † and accompanying discussion.) The CRET-based analysis of nucleic acids by hemin/Gquadruplex-modied dye-loaded NMOFs was further applied for the detection of genes and for the multiplexed analysis of the p53 and BRCA1 genes. Fig. 7(A) depicts the sensing platform for the analysis of the p53 gene. The anchor (4)-modied UiO-66 NMOFs were loaded with Fl, and hairpin hp7 was hybridized with the (4)-anchor units associated with the NMOFs. The loop domain of hp7 included the recognition sequence for p53, and the stem region of the hairpin included the G-quadruplex sequence in a caged conguration. In the presence of the p53 gene, hairpin hp7 is opened, leading to the uncaged Gquadruplex sequence, and to the formation of the hemin/Gquadruplex units. The resulting DNAzyme catalyzed oxidation of luminol by H 2 O 2 generated chemiluminescence and the CRET-guided uorescence of Fl (l em ¼ 520 nm) (Fig. 7(B)). The resulting signal is controlled by the concentrations of analyte p53, and as the concentration of p53 increases, the CRET signals are intensied. The sensing platform enabled the detection of p53 with a detection limit corresponding to 3.3 nM ( Fig. 7(C)). Similarly, the hairpin hp8 was hybridized with the (4)-modied Rh 6G-loaded NMOFs (Fig. S10(A) †). The loop domain of hp8 included the recognition sequence of the BRCA1 gene, while the stem part included the caged G-quadruplex sequence. In the presence of the BRCA1 gene, hairpin hp8 was opened, resulting in the formation of the catalytic hemin/Gquadruplex. In the presence of H 2 O 2 and luminol, the hemin/Gquadruplex catalyzed the generation of chemiluminescence, and the CRET-guided uorescence of Rh 6G (l em ¼ 550 nm) proceeded ( Fig. S10 (B) †). The CRET signals were controlled by the concentrations of the BRCA1 gene, and as they increased, the CRET signals were intensied ( Fig. S10(C) †). The method enabled the analysis of the BRCA1 gene with the detection limit corresponding to 6.7 nM. (For the multiplexed analysis of p53 and/or BRCA1 by the mixture consisting of the Fl-loaded hp7functionalized NMOFs and the Rh 6G-loaded hp8functionalized NMOFs, see ESI Fig. S11 † and accompanying discussion.) As before, we used the HCR method to amplify the CRET detection platform of the genes, and we applied the amplication method for the multiplexed analysis of the genes (Fig. 8(A), panel I and panel II). Hairpins hp9 and hp10 were hybridized, as gating units, with the (5)-modied Fl/Rh 6G-loaded NMOFs, respectively. The loop domains of hp9 and hp10 included the specic recognition sequences for the p53 or BRCA1 gene,  respectively. In the presence of the p53 or BRCA1 genes, the respective hairpins are unlocked to initiate, in the presence of hairpins hp3 and hp4, the HCR process. Hairpins hp3 and hp4 were engineered to yield, upon the HCR process, the hemin/Gquadruplex units embedded in HCR chains that, in the presence of hemin/H 2 O 2 /luminol, improve the process of CRET to the uorophores entrapped in the NMOF carriers, thereby increasing the sensitivities of the detection platforms. Fig. 8(B) panel I shows the chemiluminescence and CRET spectra generated by the Fl-loaded NMOFs, upon sensing the p53 gene, and Fig. 8(C), panel I shows the CRET spectra generated by the Rh 6G-loaded NMOFs, upon sensing the BRACA1 gene, using the HCR-amplied detection scheme. Fig. 8(B) and (C), panels II show the CRET signal-derived calibration curves corresponding to the analysis of p53 and BRCA1 genes by the Fl-and Rh 6Gloaded NMOFs. The p53 and BRCA1 genes are analyzed with detection limits corresponding to 0.33 nM and 1.67 nM, using the HCR amplied pathway. The sensitivities for sensing p53 and BRCA1 are 10-fold and 4-fold improved by the HCR amplication as compared to non-amplied sensing platform presented in Fig. 7 and S10. † As before, the HCR amplied CRET-detection platform can be used for the multiplexed analysis of the genes (Fig. S9 †). The mixture of hp9-functionalized Fl-loaded NMOFs and hp10-modied Rh 6G-loaded NMOFs was used for the multiplexed analysis of the genes. In the presence of the p53 gene, only the HCR process associated with Fl-loaded NMOFs is activated, leading to the CRET spectra of Fl ( Fig. 9(A), panel I and panel II).
In the presence of the BRCA1 gene, only the HCR process associated with Rh 6G-loaded NMOFs is activated, leading to the CRET spectra of Rh 6G (Fig. 9(B), panel I and panel II). In the presence of the two genes p53 and BRCA1, the HCR process associated with the two kinds of NMOF is activated, leading to the CRET-stimulated signals corresponding to Fl (l em ¼ 520 nm) and Rh 6G (l em ¼ 550 nm) ( Fig. 9(C), panel I and panel II).

Conclusions
The present study has introduced metal-organic framework porous nanoparticles, NMOFs, as functional carriers of uorophores for the chemiluminescence resonance energy transfer (CRET)-stimulated analysis of miRNAs and genes. The integration of hairpin units that self-assemble into functional hemin/ G-quadruplex catalytic modules stimulated the generation of CRET-induced uorescence outputs of the uorophores entrapped in the NMOFs, upon sensing the miRNAs or the genes. By the integration of different uorophores (energy acceptors) in the NMOFs, the multiplexed analysis of two different miRNAs or different genes was achieved. Furthermore, by the appropriate tailoring of the hairpins, caging the NMOFs, the amplied sensing of miRNAs or genes was demonstrated by coupling the hybridization chain reaction (HCR) to the sensing events occurring on the NMOFs. The CRET-induced sensing of analytes by the hairpin-functionalized NMOFs can be extended to other analytical targets, e.g. the detection of aptamer-ligand complexes. Beyond the signicance of the uorophore-loaded hairpin-functionalized NMOFs for sensing, the results are important because they introduce the concept of modifying and caging NMOFs by hairpin-responsive gates. Such functional NMOFs would be of signicance for the triggered release of loads (drugs) entrapped in the NMOFs.

Conflicts of interest
There are no conicts to declare.