Development of an Iridium(III) Complex as a G-Quadruplex Probe and

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Development of an iridium(III) complex as a G-quadruplex probe and its application for the G-quadruplexbased luminescent detection of picomolar insulin Modi Wang, Wanhe Wang, Tian-Shu Kang, Chung-Hang Leung, and Dik-Lung Ma Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04064 • Publication Date (Web): 26 Nov 2015 Downloaded from http://pubs.acs.org on December 2, 2015

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Analytical Chemistry

Development of an iridium(III) complex as a G-quadruplex probe and its application for the G-quadruplex-based luminescent detection of picomolar insulin Modi Wang,† Wanhe Wang,† Tian-Shu Kang,§ Chung-Hang Leung§ and Dik-Lung Ma,*†‡

†Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China

§State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China

‡Partner State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Hong Kong, China * Corresponding author: Dr. Dik-Lung Ma, E-mail: [email protected], Fax: (+852) 3411-7348, Tel: (+852) 3411-7075.

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Abstract In this study, an unreported Ir(III) complex 1 was identified by screening as a versatile G-quadruplex probe. It exhibited highly selective response for different G-quadruplex DNA over double strand, single strand and triplex DNA. Compared with the organic G-quadruplex probe thioflavin T, complex 1 displays a longer lifetime, a larger Stokes shift, comparable G-quadruplex/ssDNA enhancement ratios and higher G-quadruplex/triplex DNA enhancement ratios. In consideration of the encouraging G-quadruplex probe performance of complex 1, we employed 1 to develop a G-quadruplex-based detection system for the detection of insulin as a “proof-of-principle” concept. We also demonstrate an optimization process that enhanced the sensitivity of this sensing assay. Compared to previously reported methods, our “mix-and-detect” detection methodology is

easy operated, quick and cost-effective.

Detection limit as low as 80 pM for insulin can be achieved by this sensing approach, with a linear relationship between luminescence intensity and insulin concentration established from 80 pM to 20 nM. Moreover, this assay could work effectively in diluted human serum.


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INTRODUCTION Insulin belongs to a kind of peptide hormone that plays a key role of regulating carbohydrate and fat metabolism, in order to maintain blood glucose levels within a narrow range.1 Insulin levels are also a predictor of diabetes caused by insulinoma and trauma.2 Moreover, numerous epidemiological and pre-clinical studies have indicated that insulin levels are related to the development and progression of several types of cancer.3-5 Therefore, the accurate detection of insulin is of high importance for clinical diagnostics. Guanine (G)-quadruplexes are secondary DNA structures that can be formed from G-rich DNA sequence.6 G-quadruplexes have been recognized as effective signal-transducing unit for the construction of G-quadruplex-based sensing assay due to their extensive structural polymorphism.7-16 In a typical G-quadruplex-based sensing platform, the addition of an analyte triggers the conformational switching of a designed DNA sequence, usually in ssDNA or dsDNA form, into a G-quadruplex structure. A G-quadruplex probe can then be used to transduce the conformational change of the designed DNA sequence into a luminescent response. Many organic molecules have been reported as G-quadruplex probes, including crystal violet (CV),17 thiazole orange (TO),18 protoporphyrin IX (PPPIX),19,20 thioflavin T (ThT)21-24 and their analogues.25 These probes exhibit high fluorescence enhancement with addition of G-quadruplex DNA over other forms of DNA, although their selectivity can be influenced by other factors such as salt concentration and buffer composition.25



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On the other hand, luminescent transition metal complexes have attracted tremendous studies about the discovery of light-emitting materials26 and the employment in sensing and imaging.27-29 Transition metal complexes reveal many advantages which make them possible for sensing applications: (i) compared with organic fluorophores, metal complexes are generally more easily to synthesize without labour-intensive synthetic protocols; (ii) their photophysical properties and interactions with biomolecules can be readily tuned by variation of the co-ligands;30-34 (iii) the large Stokes shifts of metal complexes can reduce self-quenching; and (iv) their long lifetimes allows their phosphorescence to be monitored in high auto-fluorescence sample through utilizing time-resolved spectroscopy. In particular, Ir(III) complexes have received recent interest as selective G-quadruplex-binding probes.35 Compared to d8 square planar Pt(II) complexes, the octahedral structure of Ir(III) complexes can provide an alternative scaffold for G-quadruplex binding while also avoiding intercalation. Meanwhile, Ir(III) also generally have longer lifetimes and higher quantum yields compared to Ru(II). Finally, unlike most Ru(II) G-quadruplex probes, the Ir(III) complexes developed by our group have a wide range of emission wavelengths (from green to red) based on the nature of the co-ligand used. Nucleic acid aptamers, which are single-stranded oligonucleotides screened in vitro through the method of SELEX (systematic evolution of ligands by exponential enrichment), have been developed for a vast range of analytes.36-39 Comparing with antibodies, nucleic acid


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aptamers have several advantages render them important in diagnostic tools. The chemical synthesis of nucleic acid aptamers is relatively inexpensive and accurate with simple protocol, while compared to antibodies, it is usually more stable at wider range of temperature and pH values. We report herein an unreported Ir(III) complex 1 which was identified by screening, as a versatile G-quadruplex probe. Complex 1 displayed highly selective response for different sequences of G-quadruplex DNA over dsDNA, ssDNA and triplex DNA. Encouraged by the selectivity of complex 1, we employed 1 for the development of a G-quadruplex-based sensing assay for the detection of insulin as a “proof-of-principle” concept. We also demonstrate an optimization process that enhanced the sensitivity of this G-quadruplex-based sensing platform. The proposed mechanism of this sensing platform is outlined in Scheme 1. The

insulin

aptamer

(green

line)-containing

AG3AG3CGCTG3C6G3G2TG2TG8T2G2TAG3TGTCT2C-3′),

DNA which

ON1 includes

(5′a

G-quadruplex-forming sequence (black line) as well at the 5′-end of DNA, is firstly hybridized with a partially antisense DNA strand (ON2, 5′-AC8AC2AC5G6C3AGC-3′, blue line), to form a double substrate. Upon the addition of insulin into the ON1-ON2 duplex substrate, it will lead to form an insulin-aptamer complex, causing the dissociation of ON1 from ON2. After that, due to the stabilizing effect of K+ ions, the released 5′-terminus sequence of ON1 will undergo conformational change into a G-quadruplex motif. The



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induced G-quadruplex structure is then identified by the G-quadruplex-selective Ir(III) complex showing a luminescence enhancement and making the system able to serve as a sensitive probe for insulin detection. To the best of our knowledge, no luminescent G-quadruplex-based insulin sensing platform has been reported in the literature up to now.

Scheme 1. Schematic diagram illustrating the sensing mechanism of the G-quadruplex-based detection platform for insulin using the G-quadruplex-selective iridium(III) complex 1.

EXPERIMENTAL Detection of insulin ON1 (100 µM) and ON2 (100 µM) DNA were mixed in buffer (20 mM Tris, pH 7.2). The solution was gradually heated to 95 °C and kept for 10 min, after which, the temperature was decreased to about 25 °C at the speed of 0.1 °C/s. To confirm the formation of initiate duplex substrate, the treated solution was then further incubated at room temperature for 1 h. The annealed DNA sample was stored at –20 °C before use. The indicated concentration of insulin and ON1-ON2 duplex were added to a 30 µL solution of Tris buffered solution (20 mM


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Tris-HCl, pH 7.2). The mixture was incubated at 30 °C for 1 h. The samples were added to 469 µL Tris-HCl buffer (20 mM Tris, 53.3 mM KCl, pH 7.2). Finally, 1 µL of complex 1 (1 µM in final volume) was added to the mixture (20 mM Tris, 50 mM KCl, 0.5 µM duplex, 1 µM complex 1, pH 7.2). Emission spectra of complex 1 were measured in the 550−750 nm range using an excitation wavelength of 350 nm.

RESULTS AND DISCUSSION

Identifying the G-quadruplex-selectivity of iridium(III) complexes. In this work, we first synthesized a random library of ten Ir(III) complexes (1–10, Figure 1, S1) and investigated their luminescent response towards various DNA conformations, such as

G-quadruplex,

ssDNA and dsDNA (Table S1). Among these ten Ir(III) complexes, complex 1 which contains the N^N donor 2,2'-biquinoline (biq) and the C^N ligand 3-methyl-2-phenylpyridine, displayed the highest luminescent response for G-quadruplex DNA over ssDNA and dsDNA. A luminescent enhancement fold-change of ca. 5.4 and 5.3-fold was observed for complex 1 for G-quadruplex DNA over ssDNA and dsDNA, respectively (Figure 1). Based on these results, a brief structure-activity relationship can be determined. The N^N ligand of 8 contains more benzyl groups than the 2,2'-bipyridine (bpy) N^N ligand of complex 10, indicating that the size of the N^N ligand may be an important determinant for lighting up the G-quadruplex. However, excessively bulky N^N ligands such as 4,4'-dinonyl-2,2'-bipyridine (in 9), which



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has two nonyl groups attached to the bpy motif, appear to detract from G-quadruplex. While fluoro (in 3) and aldehyde (in 4) groups also displayed the detraction from G-quadruplex.

Figure 1. Diagrammatic bar array representation of the luminescence enhancement selectivity ratio of complexes 1–10 upon the addition of c-kit87up G-quadruplex over ssDNA or dsDNA.

Investigation of G-quadruplex probe 1. We then investigated the photophysical property of complex 1. The lifetime of complex 1 was 4.369 µs, which is typical for phosphorescent metal complexes, compared to only ca. 10 ns for the organic G-quadruplex probe ThT.21 The result highlights the possibility of taking advantage of the long-lived phosphorescence of complex 1 to circumvent interference from short-lived autofluorescence in complex biological samples. The maximum emission wavelength of 1 was 630 nm with an excitation maximum of 350 nm. Moreover, the Stokes shift of 280 nm for 1 which is 4.3-fold larger than that of ThT.35


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We next studied the luminescence of the novel complex 1 towards various kinds of DNA. The luminescence enhancement of complex 1 was significantly increased upon the addition of different G-quadruplex DNA, especially the c-kit-type G-quadruplexes, such as c-kit87up, c-kit1 and c-kit2 (Figure 2b, S2). A ca. 10.4-fold luminescent enhancement was observed for complex 1 in the presence of 5 µM of c-kit87up, while ca. 9.1-fold and 8.4-fold luminescent enhancements were observed for c-kit1 or c-kit2 G-quadruplex DNA, respectively. The c-kit G-quadruplex sequences are located in the promoter region of the human c-kit oncogene, which is a potential therapeutic target for the treatment of gastrointestinal tumors due to its role in the development of mast cells, melanocytes and hematopoietic stem cells.40,41 Complex 1 also displayed ca. 7-fold luminescent enhancements towards c-myc G-quadruplexes (c-myc, Pu27 and Pu22). Meanwhile, 6.3-fold and 7.9-fold luminescent enhancements were observed upon addition of human telomeric G-quadruplex 22AG and HTS, respectively, while a 8.8-fold enhancement was observed for the Plasmodium telomeric G-quadruplex. In contrast, in the presence of 5 µM of ssDNA, dsDNA and triplex DNA, no significant change in the luminescence of complex 1 was detected.

As a G-quadruplex-based detection platform generally exploits the switching of DNA from

ssDNA

or

dsDNA

to

G-quadruplex,

the

high

G-quadruplex/ssDNA

or

G-quadruplex/dsDNA luminescence enhancement ratios of 1 can increase the sensitivity of the detection platform. The c-kit87up/ssDNA enhancement ratio of complex 1 was 5.4, and



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7.6 for the c-kit87up G-quadruplex with an extended loop. This selectivity is comparable to the classic organic G-quadruplex probe ThT (ca. 8 for 22AG/ssDNA).35 Interestingly, complex 1 only showed a 1.3-fold enhancement towards triplex DNA, resulting in a G4/triplex enhancement ratio of 7.7. On the other hand, ThT showed a relative high luminescence enhancement (ca. 32-fold) towards triplex DNA with a G4/triplex ratio of 3.7.35 This may be attributed to the octahedral structure of Ir(III) complex which prevents intercalation into the groove of triplex DNA.

Figure 2. (a) Chemical structure of complex 1. (b) Fold change of complex 1 (1 µM) in the presence of 5 µM of ssDNA, dsDNA, triplex or various G-quadruplexes in 50 mM K+. (c) G4-FID mapping of complex 1. (d) Melting profile of F21T G-quadruplex DNA (0.2 µM) in the absence and presence of 1 (3 µM). (e) Melting profile of F10T dsDNA (0.2 µM) in the absence and presence of 1 (3 µM). (f) Melting profile of F21T G-quadruplex DNA (0.2 µM) in the absence and presence of 1 (3 µM) and ds26 (10 µM) or ssDNA (10 µM).


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Complex 1 showed weak luminescence emission in aqueous buffered solution containing 0.2% organic co-solvent, but becomes emissive upon binding to G-quadruplex DNA. We anticipate that the luminescence of complex 1 is owing to the shielding of the metal centre from solvent interactions upon G-quadruplex binding, thereby suppressing non-radiative decay of the excited state and enhancing triplet metal-to-ligand charge-transfer (3MLCT) emission.42-44 As a result, complex 1 only shows strong luminescence with G-quadruplex because it does not bind to other DNA structures.

For further evaluation of complex 1, we examined the quantum yield of complex 1 in different solvent environments. The quantum yield of complex 1 in aqueous buffered solution was 0.007, while a higher value of 0.050 was recorded in acetonitrile. This indicates that the luminescence of complex 1 is sensitive towards the solvent environment. Moreover, G-quadruplex fluorescent intercalator displacement (G4-FID) assays and fluorescence resonance energy transfer (FRET) melting assays were carried out to investigate the selective G-quadruplex-binding of complex 1. The G4-FID assay is a simple and convenient method to investigate the DNA binding of ligands. Binding of ligands to the G-quadruplex will displace the dye TO, resulting in a loss of fluorescence. The results showed that upon addition of 2 µM 1, about 50% of TO was displaced from G-quadruplex DNA, while only 10% of TO was displaced from dsDNA even at 5 µM of 1 tested (Figure 2c, S3). The experiment data suggests that 1 binds more strongly to G-quadruplex DNA compared to dsDNA. Additionally,



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in the FRET melting assay, 3 µM of complex 1 increased the melting temperature (∆Tm) of the F21T G-quadruplex by about 5 °C (Figure 2d), but increased the ∆Tm of F10T dsDNA by only 1.1 °C under the same conditions (Figure 2e). This indicates that 1 has a ca. 5-fold stronger stabilizing effect on G-quadruplex DNA compared to dsDNA. Moreover, in the presence of 50 times higher concentration of unlabeled dsDNA (ds26) or ssDNA as competitive species, it does not significantly influence the stabilizing effect of 1 towards the F21T G-quadruplex (Figure 2f), further confirming the G-quadruplex-selectivity of 1. The experiment data suggests that complex 1 is sensitive to changes in the solvent environment, and suggest that the strong luminescence of 1 upon the addition of G-quadruplex only is due to a change in the local environment of complex 1 upon selectively binding to G-quadruplex DNA.

G-quadruplex structures can be influenced by the presence and nature of stabilizing ions. Therefore, we also examined the G-quadruplex selectivity of complex 1 in Na+ ions. Encouragingly, the IG-quadruplex/IssDNA, IG-quadruplex/IdsDNA and IG-quadruplex/ItriplexDNA luminescent enhancement ratios were similar to the result in K+ ions (Figure S4).

It is interesting that complex 1 shows a weak luminescence towards the thrombin-binding aptamer (TBA). It has been previously reported that TBA ligands prefer end-stacking binding,45 suggesting that complex 1 may instead bind to the loop regions of the other G-quadruplexes. To examine if the loop is involved with the interaction between 1 and


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G-quadruplex DNA, we examined the luminescence signal of complex 1 against G-quadruplexes with various loop length. We explored G-quadruplex sequences containing a 3′ side loop (5′-AG3AG3CGCTG3CnG3-3′) containing different sizes of loop varying from 1 to 12 nucleotides (n = 1, 2, 3, 4, 5, 6, 8, 10, 12). We found that the luminescence signal of complex 1 gradually enhanced with longer loop sizes (n = 2 to 6), reaching a maximum fold enhancement at about 6 bases (Figure 3, S5). An exception to this trend was a G-quadruplex structure with a 1-nt loop, which showed relatively high luminescent enhancement, potentially due to the appearance of an intermolecular G-quadruplex. This experiment result outlines that G-quadruplex loop may play a crucial role in the G-quadruplex-1 binding.

Figure 3. Luminescence intensity of complex 1 with G-quadruplexes of different loop size (in nucleotides) at 3′ side loop.

Luminescent detection of insulin in aqueous buffered solution. Given the promising G-quadruplex-selective luminescent response displayed by complex 1, we decided to utilize 1 as a G-quadruplex-selective probe to developed a label-free luminescent sensing system for insulin in aqueous solution. To perform the assay, insulin (1 µM) was added into a solution



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containing ON1 and ON2 DNA (0.5 µM). After incubation at 25 °C for 60 min, 50 mM K+ ions was added to the solution, followed by the addition of complex 1 (1 µM). Encouragingly, we found that the luminescence signal of complex 1 was increased upon the addition of insulin (Figure 4a). However, no luminescence enhancement of complex 1 was obtained for the situation in the absence of ON1 and ON2 DNA (Figure 4b, S6), pointing out that the increasing luminescence signal of complex 1 was not caused by the direct interaction between 1 and insulin. We envisage that the increasing luminescence signal of the system was resulted from the selective binding of insulin to the aptamer, which induces the dissociation of ON1 from ON2, allowing the guanine-rich sequence of ON1 to change into a G-quadruplex structure with the stabilization of K+ ions that is eventually identified by 1. To demonstrate the mechanism of this approach, we designed a mutant DNA sequence (ON1m, 5′-AGT2AT3CGCTT3C6T3G2TT2TG3T5T2T2TAT3TGATT2C-3′) that cannot bind with insulin and obtain a G-quadruplex due to mutations in both the aptamer sequence as well as the G-quadruplex-forming motif. Consistent with our expectation, by utilizing the mutant DNA in system, the luminescence intensity of complex 1 didn’t show any enhancement upon the addition of insulin, suggesting that the specific aptamer-insulin interaction and subsequent G-quadruplex folding is required for an increased luminescence signal to insulin (Figure 4c, S7). The DNA conformational transition induced by insulin was confirmed by circular dichroism (CD) spectroscopy (Figure 4d). In the presence of insulin, the spectrum changes to


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show a positive band at about 260 nm, and a slight negative peak at about 235 nm.46,47 Above all, these experiment results indicates that the increasing luminescence signal of the system came from the specific binding of 1 with the G-quadruplex motif, which is produced by the dissociation of the duplex DNA substrate by insulin.

Figure 4. (a) Emission spectra of the system ([1] = 1 µM, [ON1-ON2 duplex substrate] = 0.5 µM, [K+] = 50 mM) in the presence or absence of insulin (1 µM). (b) Enhanced luminescence signal of the system in response to insulin (1 µM) in the presence or absence of ON1-ON2 duplex substrate (0.5 µM). (c) Relative luminescence response of the system using ON1, ON2 and mutated ON1, ON2. (d) CD spectrum of 0.5 µM ON1-ON2 duplex substrate in the absence and presence of 1 µM of insulin. (e)



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Emission spectrum of the system upon the addition of various concentrations of insulin. (f) Linear plot of enhanced luminescence intensity at λ = 636 nm vs. insulin concentration for ON1-ON2 (g) Relative luminescence increase of the system upon the addition of 1 µM insulin or 5 µM other interfering substances. (h) Linear plot of enhanced luminescence intensity at λ = 636 nm vs. insulin concentration for ON1-ON212

To obtain the optimal performance of the sensing platform, we investigated the effect of several parameters that may influence the performance of the assay. Firstly, it was observed that the relative luminescence signal of the system was affected by the concentration of complex 1, with a maximal response obtained at 1 µM of complex (Figure 5a, S8). Secondly, as K+ ions play a key role in the G-quadruplex folding, the effect of K+ ion concentration on the luminescence intensity of the system was also investigated. It was observed that 50 mM of K+ ions offered the highest luminescence enhancement for the detection of same concentration of insulin compared to 10, 20 or 100 mM of K+ ions (Figure 5b, S9). Furthermore, we observed that the performance of the system was the best when the concentration of ON1-ON2 duplex substrate was 0.5 µM (Figure 5c, S10). After optimization, we examined the luminescence signal of the system upon addition of increasing concentrations of insulin. The luminescence intensity of complex 1 was increased as more insulin was added (Figure 4e). The system displayed a ca. 3.4-fold enhancement in luminescence at [insulin] = 5 µM, with a linear range of detection for insulin from 20 to 500


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nM (Figure 4f). Furthermore, this approach has achieved a detection limit of 20 nM for insulin at signal-to-noise ratio (S/N) of 3.

Figure 5. (a) Relative luminescence increase of the system with various concentrations of 1 (0.25, 0.5, 1, 2 and 3 µM). (b) Relative luminescence increase of the system with various concentrations of KCl (10, 20, 50 and 100 mM). (c) Relative luminescence increase of the system with various concentrations of ON1-ON2 duplex substrate (0.25, 0.5, 1 and 2 µM). Unless specifically stated, the concentration of complex 1 was 1 µM, the concentration of ON1-ON2 duplex substrate was 0.5 µM and the concentration of KCl was 50 mM.

Moreover, we also constructed a similar detection assay using the traditional organic G-quadruplex probe, ThT, for comparative testing. In an emission titration experiment, the luminescence intensity of ThT (490 nm) was increased with higher concentrations of insulin (Figure S11). The ThT system exhibited a detection limit of 70 nM, which was less sensitive than that of the detection platform utilising the luminescent iridium(III) complex 1. This result demonstrates the importance of identifying or developing the most suitable G-quadruplex probes to be used for specific types of detection platforms. In this regard, iridium(III)



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complexes represent attractive scaffolds for the exploration of G-quadruplex-binding ligands as they are easily synthesised and exhibit properties that can be readily tuned by adjustment of their auxiliary ligands.

The selectivity of our assay for insulin was tested by studying the luminescent behavior of the system to other interfering substances including metal ions (K+, Ca2+, Mg2+, Fe3+), amino acids (glutamic acid (Glu) and glycine (Gly)), peptides (glutathione (GSH)) and proteins (bovine serum albumin (BSA)). The results displayed that only insulin could significantly enhance the luminescence signal of the sensing system (Figure 4g, S12). No significant luminescent enhancement was obtained in the presence of the other substances. These results suggest that the system shows high selectivity for insulin compared to other substances, which originates presumably from the specific binding between insulin and its aptamer.

In our design, the G-quadruplex-forming sequence in ON1 is initially masked due to hybridization of ON1 to its partially complementary DNA sequence (ON2). If only one of the G-tracts are hybridized, the duplex-to-G-quadruplex conversion may occur when K+ ions are added even in the absence of insulin, thus resulting in a high background signal. On the other hand, if three or four G-tracts of ON1 are hybridized, the initial duplex substrate may be too stable and hence resistant to dissociation induced by insulin, which would decrease the sensitivity of the assay. To optimize the sensitivity limit of this platform, potentially towards


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the concentration range of insulin in human blood, the complementary sequence ON2 was carefully optimized. The complementary sequence ON2 hybridizes using 15 bases with the G-quadruplex-forming sequence in ON1. We therefore also investigated ON2 derivatives that hybridize using 9, 12, 15, 18, 21 or 24 bases. We observed that the system with 18 bases showed comparable performance to the system with 15 bases. However, the systems with 21 and 24 hybridized bases show a large decrease in luminescent enhancement in the presence of the same concentration of insulin. We attribute this result to the enhanced stability of the initial duplex substrate with 21 or 24 hybridized bases, which resists dissociation induced by insulin, hence decreasing the sensitivity of the assay. Interestingly, the system with 12 hybridized bases showed higher luminescence enhancement for the detection of same concentration of insulin compared to 9 and 15 bases. The lower enhancement for the system with 9 hybridized bases could be due to duplex-to-G-quadruplex conversion even in the absence of insulin, thus resulting in a high background signal. Therefore, the ON2 complementary

DNA

sequence

with

12

bases

(ON212)

hybridized

with

the

G-quadruplex-forming sequence in ON1 was employed (Figure S13). Encouragingly, after optimizing the ON2 sequence, the ON1-ON212 system displayed a linear relationship established between luminescence intensity and insulin concentration from 80 pM to 20 nM (Figure 4h, S14). Moreover, this approach has achieved a detection limit of 80 pM for insulin at signal-to-noise ratio (S/N) of 3.



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Application of the insulin sensing platform in biological samples. We next examined the suitability of the assay to detect insulin in a biological matrix. We performed the detection assay in a sensing system containing 0.5% (v/v) human serum. We observed that the 1/DNA system showed enhanced luminescence intensity in diluted human serum as the concentration of insulin was increased (Figure S15). This result verifies that this approach could potentially be further constructed for insulin detection in biological samples.

CONCLUSIONS

In conclusion, an unreported Ir(III) complex 1 was identified by screening as a versatile G-quadruplex probe. Complex 1 showed highly selective response for different G-quadruplex than dsDNA, ssDNA and triplex DNA. Compared with the organic G-quadruplex probe thioflavin T, complex 1 displays a longer lifetime, a larger Stokes shift, comparable G-quadruplex/ssDNA

enhancement

ratios

and

higher

G-quadruplex/triplex

DNA

enhancement ratios. We thus employed 1 to demonstrate the “proof-of-principle” concept of a G-quadruplex-based defection assay for insulin detection. We also have demonstrated that the optimization of the oligonucleotide sequence can increase the sensitivity of the assay. Compared to previously reported methods, our “mix-and-detect” detection methodology is simple, quick and cost-effective. Detection limit as low as 80 pM for insulin can be achieved by this sensing approach, with a linear relationship between luminescence intensity and insulin concentration established from 80 pM to 20 nM. Furthermore, this sensing platform


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could work effectively in diluted human serum. We envision that our versatile G-quadruplex probe complex 1 and this label-free G-quadruplex-based switch-on luminescent detection assay for insulin could aid the study of clinical diagnosis.

Acknowledgments This work is supported by Hong Kong Baptist University (FRG2/14-15/004), the Health and Medical Research Fund (HMRF/13121482 and HMRF/14130522), the Research Grants Council (HKBU/201811, HKBU/204612 and HKBU/201913), the French National Research Agency/Research Grants Council Joint Research Scheme (A-HKBU201/12), State Key Laboratory of Environmental and Biological Analysis Research Grant (SKLP-14-15-P001), National Natural Science Foundation of China (21575121), Guangdong Province Natural Science Foundation (2015A030313816), Hong Kong Baptist University Century Club Sponsorship

Scheme

2015,

Interdisciplinary

Research

Matching

Scheme

(RC-IRMS/14-15/06), the State Key Laboratory of Synthetic Chemistry, the Science and Technology Development Fund, Macao SAR (103/2012/A3 and 098/2014/A2), the University of

Macau

(MYRG091(Y3-L2)-ICMS12-LCH,

MYRG2015-00137-ICMS-QRCM

MRG023/LCH/2013/ICMS) AUTHOR INFORMATION Corresponding Author


and


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*E-mail: [email protected]

Notes The authors declare no competing financial interest.

Additional information Competing financial interests: The authors declare no competing financial interests. Supporting Information. Detailed protocols for sample preparations and analyses. This material is available free of charge via the Internet at http:// pubs.acs.org.

References (1) Pu, Y.; Zhu, Z.; Han, D.; Liu, H.; Liu, J.; Liao, J.; Zhang, K.; Tan, W. Analyst 2011, 136, 4138-4140. (2) Gobi, K. V.; Iwasaka, H.; Miura, N. Biosens. Bioelectron. 2007, 22, 1382-1389. (3) Renehan, A. G.; Frystyk, J.; Flyvbjerg, A. Trends Endocrinol. Metab. 2006, 17, 328-336. (4) Lazarus, D. D.; Kambayashi, T.; Lowry, S. F.; Strassmann, G. Cancer. Lett. 1996, 103, 71-77. (5) Yam, D. Med. Hypotheses. 1992, 38, 111-117. (6) Mendez-Bermudez, A.; Hills, M.; Pickett, H. A.; Phan, A. T.; Mergny, J.-L.; Riou, J.-F.; Royle, N. J. Nucleic Acids Res. 2009, 37, 6225-6238. (7) Wu, L.; Qu, X. Chem. Soc. Rev. 2015, 44, 2963-2997. (8) Zhao, C.; Wu, L.; Ren, J.; Xu, Y.; Qu, X. J. Am. Chem. Soc. 2013, 135, 18786-18789. (9) Peng, Y.; Wang, X.; Xiao, Y.; Feng, L.; Zhao, C.; Ren, J.; Qu, X. J. Am. Chem. Soc. 2009, 131, 13813-13818. (10) Song, S.; Qin, Y.; He, Y.; Huang, Q.; Fan, C.; Chen, H.-Y. Chem. Soc. Rev. 2010, 39, 4234-4243. (11) Li, D.; Shlyahovsky, B.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2007, 129, 5804-5805.


Page 22 of 25

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Freeman, R.; Sharon, E.; Teller , C.; Henning , A.; Tzfati, Y.; Willner, I. ChemBioChem 2010, 11, 2362-2367. (13) Zhu, J.; Li, T.; Zhang, L.; Dong, S.; Wang, E. Biomaterials 2011, 32, 7318-7324. (14) Shimron, S.; Wang, F.; Orbach, R.; Willner, I. Anal. Chem. 2011, 84, 1042-1048. (15) Lv, L.; Guo, Z.; Wang, J.; Wang, E. Curr. Pharm. Des. 2012, 18, 2076-2095. (16) Fujita, H.; Imaizumi, Y.; Kasahara, Y.; Kitadume, S.; Ozaki, H.; Kuwahara, M.; Sugimoto, N. Pharmaceuticals 2013, 6, 1082-1093. (17) Kong, D.-M.; Guo, J.-H.; Yang, W.; Ma, Y.-E.; Shen, H.-X. Biosens. Bioelectron. 2009, 25, 88-93. (18) Lubitz, I.; Zikich, D.; Kotlyar, A. Biochemistry 2010, 49, 3567-3574. (19) Zhang, Z.; Sharon, E.; Freeman, R.; Liu, X.; Willner, I. Anal. Chem. 2012, 84, 4789-4797. (20) Li, T.; Wang, E.; Dong, S. Anal. Chem. 2010, 82, 7576-7580. (21) Mohanty, J.; Barooah, N.; Dhamodharan, V.; Harikrishna, S.; Pradeepkumar, P. I.; Bhasikuttan, A. C. J. Am. Chem. Soc. 2013, 135, 367-376. (22) Gabelica, V.; Maeda, R.; Fujimoto, T.; Yaku, H.; Murashima, T.; Sugimoto, N.; Miyoshi, D. Biochemistry 2013, 52, 5620-5628. (23) Renaud de la Faverie, A.; Guédin, A.; Bedrat, A.; Yatsunyk, L. A.; Mergny, J.-L. Nucleic Acids Res. 2014. (24) Liu, L.; Shao, Y.; Peng, J.; Huang, C.; Liu, H.; Zhang, L. Anal. Chem. 2014, 86, 1622-1631. (25) Kataoka, Y.; Fujita, H.; Kasahara, Y.; Yoshihara, T.; Tobita, S.; Kuwahara, M. Anal. Chem. 2014, 86, 12078-12084. (26) Wong, W.-Y.; Ho, C.-L. Acc. Chem. Res. 2010, 43, 1246-1256. (27) Gill, M. R.; Garcia-Lara, J.; Foster, S. J.; Smythe, C.; Battaglia, G.; Thomas, J. A. Nat. Chem 2009, 1, 662-667.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(28) Zhao, Q.; Li, F.; Huang, C. Chem. Soc. Rev. 2010, 39, 3007-3030. (29) Zhao, Q.; Huang, C.; Li, F. Chem. Soc. Rev. 2011, 40, 2508-2524. (30) Leung, K.-H.; Ma, V. P.-Y.; He, H.-Z.; Chan, D. S.-H.; Leung, C.-H.; Ma, D.-L. RSC Adv. 2012, 2, 8273-8276. (31) Li, C.; Yu, M.; Sun, Y.; Wu, Y.; Huang, C.; Li, F. J. Am. Chem. Soc. 2011, 133, 11231-11239. (32) Yu, M.; Zhao, Q.; Shi, L.; Li, F.; Zhou, Z.; Yang, H.; Yi, T.; Huang, C. Chem. Commun. 2008, 2115-2117. (33) Liu, Q.; Yang, T.; Feng, W.; Li, F. J. Am. Chem. Soc. 2012, 134, 5390-5397. (34) Liu, Q.; Yin, B.; Yang, T.; Yang, Y.; Shen, Z.; Yao, P.; Li, F. J. Am. Chem. Soc. 2013, 135, 5029-5037. (35) Renaud de la Faverie, A.; Guédin, A.; Bedrat, A.; Yatsunyk, L. A.; Mergny, J.-L. Nucleic Acids Res. 2014, 42, e65. (36) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822. (37) Tuerk, C.; Gold, L. Science 1990, 249, 505-510. (38) Imaizumi, Y.; Kasahara, Y.; Fujita, H.; Kitadume, S.; Ozaki, H.; Endoh, T.; Kuwahara, M.; Sugimoto, N. J. Am. Chem. Soc. 2013, 135, 9412-9419. (39) Hagiwara, K.; Fujita, H.; Kasahara, Y.; Irisawa, Y.; Obika, S.; Kuwahara, M. Mol. BioSyst. 2015, 11, 71-76. (40) Hulzinga, J. D.; Thuneberg, L.; Kluppel, M.; Malysz, J.; Mikkelsen, H. B.; Bernstein, A. Nature 1995, 373, 347-349. (41) Rankin, S.; Reszka, A. P.; Huppert, J.; Zloh, M.; Parkinson, G. N.; Todd, A. K.; Ladame, S.; Balasubramanian, S.; Neidle, S. J. Am. Chem. Soc. 2005, 127, 10584-10589. (42) Winkler, J. R.; Netzel, T. L.; Creutz, C.; Sutin, N. J. Am. Chem. Soc. 1987, 109, 2381-2392. (43) Wang, X.-y.; Del Guerzo, A.; Schmehl, R. H. J. Photochem. Photobiol. C: Photochem.


Page 24 of 25

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Rev. 2004, 5, 55-77. (44) Jenkins, Y.; Friedman, A. E.; Turro, N. J.; Barton, J. K. Biochemistry 1992, 31, 10809-10816. (45) Monchaud, D.; Allain, C.; Bertrand, H.; Smargiasso, N.; Rosu, F.; Gabelica, V.; De Cian, A.; Mergny, J. L.; Teulade-Fichou, M. P. Biochimie 2008, 90, 1207-1223. (46) Kypr, J.; Kejnovská, I.; Renčiuk, D.; Vorlíčková, M. Nucleic Acids Res. 2009, 37, 1713-1725. (47) Zhu, J.; Zhang, L.; Wang, E. Chem. Commun. 2012, 48, 11990-11992.

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Development of an Iridium(III) Complex as a G-Quadruplex Probe and

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