Highly fluorescent N-doped carbon dots with two-photon emission for ultrasensitive detection of tumor marker and visual monitor anticancer drug loading and delivery

Highlights

• Novel nitrogen-doped carbon dots (N-CDs) with high quantum yield (Ca. 45%) were synthesized.

• N-CDs exhibited high quality single- and two-photon fluorescence in a high depth up to 18 μm.

• N-CDs possessed ultrasensitive detection of β-glucuronidase in both intracellular and human serum samples.

• N-CDs could visually monitor the drug loading and delivery process and exhibited better cancer therapeutic effects.

Abstract

Early diagnosis and therapy are practically important to effectively prevent cancer and improve patients’ life quality. Herein, a simple and sensitive two-photon imaging fluorescent nitrogen-doped carbon dots (N-CDs) platform was constructed to detect β-glucuronidase (βG, a tumor-invasive biomarker) based on inner filter effect (IFE), to visually monitor anticancer drug loading by fluorescence resonance energy transfer (FRET), and to effectively treat cancer by chemotherapy. To build this sensing platform, novel N-CDs with strong green emission (Ca. QY = 45%) were synthesized by one-pot pyrolysis reaction, and specifically, careful structure design of the excitation bands of N-CDs well covered the absorption bands of p-nitrophenol (PNP, βG catalytic product), making it ultra-sensitive towards the activity of βG and showing good linear relationship from 1 to 15 U/L and exciting detection limit of 0.3 U/L (S/N = 3). This sensing platform has been successfully employed for βG sensing in both human serum and intracellular detection. Moreover, we also demonstrated the first application of N-CDs as a novel visual detection sensor to monitor the loading process of doxorubicin, and as a nanocarrier for cancer therapy. More importantly, the N-CDs also exhibited high quality single- and two-photon fluorescence in high depth (18 μm), enabling the N-CDs to real-time monitor the endocytosis, intracellular distribution and release of drugs, indicating significant potential for biomedicine and bioimaging applications.

Introduction

Fluorescent carbon dots (CDs) as efficient and sensitive probes and nanocarriers are gaining considerable attention for their capabilities, such as biological imaging, early diagnosis and drug delivery [1], [2], [3], [4]. CDs can compete commercially with conventional fluorescent nanomaterials because of the former’s unusual and interesting optical properties, good dispersibility, chemical stability, low toxicity and easy synthesis [5], [6]. These advantages enable CD-based fluorescent sensors to detect ions (e.g., Hg²⁺, Cu²⁺, Ag⁺, and Fe³⁺) [7], [8], [9], [10], pH [11], and molecular substances [12] by monitoring changes in fluorescence signals. These advantages endow CDs with the ability to act as a nanocarrier for intracellular imaging and drug tracing. Nitrogen-doped CDs (N-CDs) can reportedly enhance photoluminescence [13]. Thus, developing N-CDs to expand fluorescent CD application in fluorescent probes and drug tracing is necessary.

In cancer treatment, the early detection of tumor-invasive biomarkers contributes to the diagnosis and early treatment of malignancies and leads to higher survival opportunities for cancer patients. β-Glucuronidase (βG) is one of the lysosomal glycosides that can catalyze the degradation of the extracellular matrix of normal and cancer tissues and glycosaminoglycan on the cell membranes [14]. According to earlier reports, the increased activities of βG can induce several cancers, such as liver cancer [15], colon carcinoma [16], renal carcinoma [17], and neoplasm of the bladder [18]. The over-expression of βG in the serum was also closely related to some pathological conditions, including renal diseases, epilepsy, urinary tract infection, larynx, and breast [19], [20], [21] Therefore, the specific and sensitive determination of βG is important. However, the determination method needs further study. Recently, fluorogenic substrates [22], colorimetric method [23], and chemosensor with organic fluorescent probes [15] were the most commonly used quantitative detection methods. Although these methods had their own characteristics and gave different insights for βG sensing, they possess inherent disadvantages, including low detection sensitivity, poor water solubility and photostability, poor repeatability, complicated synthesis process, surface modification and high cost. Hence, developing a novel, specific, and sensitive fluorescent probe is necessary to detect βG with high sensitivity, selectivity, operability and low cost. Recently, the inner filter effect (IFE) has emerged as an effective method for sensor design, it bases on the mechanism that when the absorber’s absorption spectra overlap with the fluorophore’s excitation or emission spectra in the detection system [24], [25]. IFE can transform the absorption responses into fluorescent signals; [26] it exhibits an enhanced sensitivity compared with other fluorescence-sensing mechanisms, such as electronic energy transfer (EET), fluorescence resonance energy transfer (FRET), and intramolecular charge transfer (ICT), because IFE does not need surface modification of the fluorescence material or the covalent linking between the receptor and fluorophore. However, screening out a suitable absorber fluorophore pair is still a great challenge due to limitations of fluorophores.

Herein, we designed and prepared a novel N-CDs with bright green emission and which gave a high fluorescence yield of about 45% using a one-pot pyrolysis strategy. The N-CDs with nitrogen groups has been reported to give excellent optical properties and substantially expand their novel applications [27]. Therefore, we developed a facile and reliable IFE-based N-CDs fluorescence sensor platform for βG-activity sensing and the N-CDs were directly used as the fluorophore (Scheme 1). 4-Nitrophenyl-β-D-glucuronide (PNPG) was selected as the substrate of βG, and p-nitrophenol (PNP) as a βG reaction product was adopted as the absorber. Interestingly, the absorption wavelength of PNP was well-overlapped with the excitation wavelength of N-CDs. Therefore, the excitation of N-CDs was weakened due to the competitive absorption between N-CDs and PNP, leading to remarkable quenching of N-CDs. The proposed sensing strategy was applied to detect βG activity in serum samples and exhibited good applicability. This sensing assay provided a satisfactory accuracy of 10 μL of serum sample consumption, which was of crucial importance for clinical studies. We also investigated the possibility of βG cell imaging using the proposed fluorescence detection strategy for the first time, and this sensing platform exhibited high sensitivity and imaging effects toward βG detection in living cells [28]. Therefore, the developed fluorescence assay was proven to have many merits, such as simplicity, low toxicity, high selectivity and superior sensitivity, which exhibited broad prospect in cancer early-stage diagnosis.

Considering the unique physicochemical and luminescent properties, N-CDs were also explored as a novel nanocarrier for tumor therapy, as well as for cellular imaging and drug tracing. This drug delivery system was constructed by attaching doxorubicin (DOX), a universal anticancer drug in the clinical treatment, to N-CDs via π-π stacking interaction [29], leading to an emission intensity of N-CDs decreased based on a FRET mechanism [30] and allowing us to visually monitor the loading process. When DOX-loaded N-CDs were taken up into tumor cells, the DOX was released from the N-CDs surface, resulting in the fluorescence recovery of N-CDs and giving rise to efficient killing of cancer cells [31]. More importantly, the N-CDs also exhibited strong two-photon fluorescence upon excitation at the near-infrared region (800–1100 nm) and possess the advantages of enhanced tissue penetration with a safer treatment [32], [33], [34]. Compared to single-photon fluorescence imaging (SPFI), the two-photon fluorescence imaging (TPFI) was highly desirable for biomedical imaging due to its low cell damage, large imaging depth, high resolution, and three-dimensional imaging capability [35], [36]. High-quality single- and two-photon cellular imaging endows N-CDs with the capability to monitor endocytosis, drug-release, and intracellular distribution in real-time, indicating its significant potential in bioimaging and cancer therapy.