Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

Isothermal Amplification of Nucleic Acids

1.1. Cancer and Early Detection
Cancer is the second most common cause of death in the United States, trailing only heart disease in incidence. Despite significant worldwide investment in research, cancer remains responsible for 1 in 4 deaths in developed countries.1 Globally, over 14 million cancer diagnoses were reported in 2012, a figure expected to increase to over 22 million cases per annum in the next two decades.2 Estimated to kill over 1/2 million U.S. citizens, and with over 1.6 million new cases predicted to be diagnosed this year,3 cancer continues to present a major, yet unmet challenge to healthcare both globally and in the United States.

Cancer emerges from our own tissues, complicating both detection and treatment methods due to the similarities between the diseased tissue and healthy tissue.4,5 Despite this fact, the mortality rate from cancer is often greatly reduced by early detection of the disease. For example, non-small-cell lung cancer is responsible for the most cancer related deaths worldwide, with patients in the advanced stages of the disease having only 5–15% and <2% 5-year survival rates for stage III and IV patients, respectively.6 In contrast, patients who start therapy in the early stages of the disease (stage I) have markedly improved survival rates, with an 80% overall 5-year survival rate.6 Consequently, early diagnosis is essential to improving cancer patient prognosis.

At present, clinical detection of cancer primarily relies on imaging techniques or the morphological analysis of cells that are suspected to be diseased (cytology) or tissues (histopathology). Imaging techniques applied to cancer detection, including X-ray, mammography, computed tomography (CT), magnetic resonance imaging (MRI), endoscopy, and ultrasound, have low sensitivity and are limited in their ability to differentiate between benign and malignant lesions.7,8 While cytology, such as testing for cervical cancer via a Pap smear or occult blood detection, may be used to distinguish between healthy and diseased cells or tissues, it is not effective at detecting cancer at early stages. Similarly, histopathology, which generally relies on taking a biopsy of a suspected tumor, is typically used to probe the malignancy of tissues that are identified through alternative imaging techniques, such as CT or MRI, and may not be used alone to detect cancer in its early stages. As such, the development of assays and methods for early detection of cancer, before the disease becomes symptomatic, presents a major challenge.

Recent research within the field of nanotechnology has focused on addressing the limitations of the currently available methods for cancer diagnosis. Certain nanoparticle probes possess several unique properties that are advantageous for use in the detection of cancer at the early stages. In this review, we will discuss the advances in the development of nanoparticle-based methods for the detection of cancer by fluorescence spectroscopy. We will divide this topic into three categories: techniques that are designed for (1) the detection of extracellular cancer biomarkers, (2) the detection of cancer cells, and (3) the detection of cancerous tissues in vivo. We will discuss these strategies within the context of the nanoparticle probe used as well as the recognition moieties applied in each approach. Ultimately, the translation of these methods from the laboratory to the clinic may enable earlier detection of cancer and could extend patient survival through the ability to administer therapeutic treatment in the early stages of the disease.

While this review provides a comprehensive overview of the nanoparticle probes that are used to detect cancer in vitro and in vivo through fluorescence, there are several other relevant reviews that may be of interest to our readers, who may refer to the references for more generalized reviews of nanomaterials used for diagnostics and therapy,9–12 or more detailed insight into the specific types of nanoparticle probes (i.e., quantum dots,13 gold nanoparticles,14,15 upconversion nanoparticles,16 polymer dots,17,18 silica nanoparticles,19 polymeric nanoparticles, 20 etc.) for cancer diagnosis.

Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence

Lanthanide ion (Ln(3+))-based upconversion nano/micromaterials that emit higher-energy visible light when excited by low-energy NIR light have aroused considerable attention in the forefront of materials science and biomedical fields, which stems from their unique optical and chemical properties including minimum photodamage to living organisms, low autofluorescence, high signal-to-noise ratio and detection sensitivity, and high penetration depth in biological or environmental samples. Thus, Ln(3+)-based upconversion materials are rising new stars and are quickly emerging as potential candidates to revolutionize novel biomedical applications. In this review article, we mainly focus on the recent progress in various chemical syntheses of Ln(3+)-based upconversion nanomaterials, with special emphasis on their application in stimuli-response controlled drug release and subsequent therapy. Functional groups that are introduced into the stimuli-responsive system can respond to external triggers, such as pH, temperature, light, and even magnetic fields, which can regulate the movement of the pharmaceutical cargo and release the drug at a desired time and in a desired area. This is crucial to boost drug efficacy in cancer treatment while minimizing the side effects of cytotoxic drugs. Many multifunctional (magnetic/upconversion luminescence and porous) composite materials based on Ln(3+) have been designed for controlled drug delivery and multimodal bioimaging. Finally, the challenges and future opportunities for Ln(3+)-based upconversion materials are discussed.

Current advances in lanthanide ion (Ln(3+))-based upconversion nanomaterials for drug delivery.

Metal nanoclusters, composed of several to a few hundred metal atoms, have received worldwide attention due to their extraordinary physical and chemical characteristics. Recently, great efforts have been devoted to the exploration of the potential diagnostic and therapeutic applications of metal nanoclusters. Here we focus on the recent advances and new horizons in this area, and introduce the rising progress on the use of metal nanoclusters for biological analysis, biological imaging, therapeutic applications, DNA assembly and logic gate construction, enzyme mimic catalysis, as well as thermometers and pH meters. Furthermore, the future challenges in the construction of biofunctional metal nanoclusters for diagnostic and therapeutic applications are also discussed. We expect that the rapidly growing interest in metal nanocluster-based theranostic applications will certainly not only fuel the excitement and stimulate research in this highly active field, but also inspire broader concerns across various disciplines.

Metal nanoclusters: novel probes for diagnostic and therapeutic applications

Replacing antibodies with aptamers in lateral flow immunoassay.

The biosynthesis of gold nanoparticles using catclaw buttercup (Radix Ranunculi Ternati) and their stability have been reported in this paper. The aqueous catclaw buttercup was used as mild reducing agent for gold nanoparticles synthesis from HAuCl4 solutions. The influence of reaction time, temperature and mass ratio of HAuCl4/catclaw buttercup were evaluated to investigate their effects on gold nanoparticles synthesis. Under the optimized reaction parameters, the gold nanoparticles obtained are characterized by UV-vis spectrum, X-ray diffraction (XRD), EDAX technique (EDX), high resolution transmission electron microscopy (HRTEM), FTIR spectrum, anthrone-sulfuric colorimetric method, and plus Improved-Lowry Protein Assay Kit. The HRTEM images showed that the biosynthesized gold nanoparticles are mostly spherical with size range from 9-24 nm. Furthermore, it was found that the biosynthesized gold nanoparticles possessed outstanding colloid stability in aqueous solutions as a function of category and concentration of monovalent salt and pH value of the solution when compared with chemosynthetic ones with the similar size. Anthrone-sulfuric colorimetric method revealed that there is no sugar in the biosynthesized gold colloid. While Improved-Lowry tests results demonstrated that the existence of much protein in the biosynthesized gold colloid, which may played an important role in stabilization of it. Owing to their stability, biocompatibility, lower cost and so on, gold nanoparticles synthesized by this biosynthesis method show potential application prospect in optoelectronic and biomedicine.

Biosynthesis of gold nanoparticles using catclaw buttercup (Radix Ranunculi Ternati) and evaluation of its colloidal stability.

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Biosynthesis of gold nanoparticles using catclaw buttercup (Radix Ranunculi Ternati) and evaluation of its colloidal stability.