Methods of detection of β-galactosidase enzyme in living cells

Highlights

• Molecular probes have emerged to replace the previously used chromo-fluorogenic molecular probes in detection of β-galactosidase activity in vivo animal models.

• These molecular probes still present some drawbacks such as poor cellular membrane permeability, inapt excitation and emission wavelengths, and diminished chemical stability.

• NIR fluorescent probes have provided powerful tools for tracking enzyme activity with enriched sensitivity and are suitable for in vivo tracking applications.

• Some fluorescent probes still have wavelengths less than 600 nm and exhibit several in vivo application drawbacks, such as low tissue penetration and auto-fluorescence from bio-specimens.

• Recent use of nanoparticles in the controlled release of fluorophores in cells that overexpress β-galactosidase enzyme have reducing unwanted harmful side effects of the loaded molecules.

Abstract

The application of β-galactosidase enzyme ranges from industrial use as probiotics to medically important application such as cancer detection. The irregular activities of β-galactosidase enzyme are directly related to the development of cancers. Identifying the location and expression levels of enzymes in cancer cells have considerable importance in early-stage cancer diagnosis and monitoring the efficacy of therapies. Most importantly, the knowledge of the efficient method of detection of β-galactosidase enzyme will help in the early-stage treatment of the disease. In this review paper, we provide an overview of recent advances in the detection methods of β-galactosidase enzyme in the living cells, including the detection strategies, and approaches in human beings, plants, and microorganisms such as bacteria. Further, we emphasized on the challenges and opportunities in this rapidly developing field of development of different biomarkers and fluorescent probes based on β-galactosidase enzyme. We found that previously used chromo-fluorogenic methods have been mostly replaced by the new molecular probes, although they have certain drawbacks. Upon comparing the different methods, it was found that near-infrared fluorescent probes are dominating the other detection methods.

Introduction

β-Galactosidase, a glycoside hydrolase enzyme, is one of the important enzymes for digestion of lactose. It is categorized in exoglycosidase family because it plays a vital role in human body in the elimination of galactose residues from different substrates like glycoproteins, gangliosides, and sphingolipids [1,2]. It also has been proved to be a crucial enzyme as it is used as a biomarker of cell senescence and primary ovarian cancer [[3], [4], [5], [6], [7]]. Besides, it has also been used in the gene expression [8], and transcriptional regulation [9]. The concentration of β-galactosidase, an important enzyme, must be maintained in the human body. The alteration of concentration of β-galactosidase has been associated with β-galactosialidosis, morquio B syndrome [10], and ovarian cancer [11,12]. Hence, it is significant to keep track of the concentration and activity changes of β-galactosidase in situ and thereby well-define the roles of β-galactosidase in associated diseases.

To monitor the β-galactosidase concentration in vitro and in vivo, many researchers are interested in developing highly selective and sensitive methods. Several researchers have adopted different methods for the effective detection of β-galactosidase. Some of the techniques that have been used in the past are bioluminescence [13], chemiluminescence [14], magnetic resonance (MR) [15,16], single photoemission computed tomography [17], positron emission tomography (PET) [18], colorimetric [19] and fluorogenic [20,21] approaches. These methods have different advantages and disadvantages. Therefore, there is a necessity of proper categorization of the methods used (Fig. 1). The methods like, MR, positron emission tomography, single photoemission computed tomography are hampered by high cost, laborious manipulation, modest sensitivity, and invalid monitoring of β-galactosidase enzyme. Moreover, these techniques have failed to achieve real-time in situ non-destructive detection of this enzyme in the biological system.

Among these techniques, fluorescent sensors [22] have been really popular owing to their convenience, high sensitivity, simple handling procedures, inexpensive instruments, and bioimaging ability. As of now, only a few fluorescent probes for β-galactosidase have been developed, with some of them applied to monitor the enzyme activity in living cells or in tissues [[23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]]. However, most of the reported sensors are fabricated with traditional fluorophores, which suff ;ered from aggregation caused quenching (ACQ) of fluorescence in high concentration solutions or after they accumulated in cells, making the fluorescence emission much weaker as compared to that in solution [26]. Great interest to develop β-galactosidase probes without the ACQ eff ;ect for living cell or tumor tissue imaging has been perceived recently. Apart from this, most of the researchers are inclined in the development of fluorescent probes for monitoring of β-galactosidase activity due to the advantage of advanced sensitivity, less expensive instrumentation, convenient handling procedures, and convenience of bioimaging.

Herein, we have reviewed various methods used in the detection of β-galactosidase in the living cells. We have provided an overview of recent advances in the detection methods of β-galactosidase enzyme in the living cells, including the detection strategies, and approaches in human beings, plants and microorganisms as shown in Fig. 2. To our knowledge, there are not any recent reviews that have compared the different methods used in the detection of β-galactosidase in living cells. We specifically summarized recent advances in the development of enzyme detection methods. The main objective of this paper is to serve as a narrow footbridge by comparing the literary works on the different methods of β-galactosidase detection, critically analyze their reliability by showing the pros and cons of the predicted methods for the practical use. In the detection of β-galactosidase both in vivo and in vitro, we assessed the progress made in designing the molecular probes from the previously used chromo-fluorogenic probes. Most of the reported molecular probes are based on the technique of attaching the galactopyranoside residue to the specific signaling units. These probes have enriched properties as compared to the traditional technique. However, they lack proper cellular membrane permeability, have inappropriate excitation and emission wavelengths, and have diminished chemical stability. With all these information, we hope that this review paper will enable to enrich the knowledge to facilitate the monitoring of β-galactosidase activity within living cells or tissues and open the minds to the interested researcher to develop new advances in the detection of this enzyme.