AbstractMillions of people worldwid

Abstract
Millions of people worldwide live with diabetes and several millions die from it each year. A noninvasive, painless method of glucose testing would highly improve compliance and glucose control while reducing complications and overall disease management costs. To provide accurate, low cost, and continuous glucose monitoring, we have developed a unique, disposable saliva nano-biosensor. More than eight clinical trials on real-time noninvasive salivary glucose monitoring were carried out on two healthy individuals (a 2–3 h-period for each trial, including both regular food and standard glucose beverage intake with more than 35 saliva samples obtained). Excellent clinical accuracy was revealed as compared to the UV Spectrophotometer. By measuring subjects’ salivary glucose and blood glucose in parallel, we found the two generated profiles share the same fluctuation trend but the correlation between them is individual dependent. There is a time lag between the peak glucose values from blood and from saliva. However, the correlation between the two glucose values at fasting is constant for each person enabling noninvasive diagnosis of diabetes through saliva instead of blood. Furthermore, a good correlation of glucose levels in saliva and in blood before and 2 h after glucose intake was observed. Glucose monitoring before and 2 h after meals is usually prescribed by doctors for diabetic patients. Thus, this disposable biosensor will be an alternative for real-time salivary glucose tracking at any time.

Keywords
Noninvasive; Saliva; Nano-biosensor; Disposable; Glucose monitoring
1. Introduction
The International Diabetes Federation estimates 382 million people worldwide had diabetes in 2013, and the number is forecasted to reach 592 million by 2035 (a 55% increase) [1]. There were 5.1 million diabetes-related deaths globally in 2013, equaling to one death every 6 s, an 11% increase over 2011 [2]. Early diagnosis, on-time treatment and continuous management are vital to patients’ life quality and to avoid complications such as circulatory problems, kidney failure, heart disease, stroke, and blindness [3] and [4]. Current practices for diabetes management rely on monitoring blood glucose levels. Blood glucose measurements are required to determine insulin dosage and to detect abnormal glucose levels indicating illnesses, dietary changes, or adverse medication responses. These intrusive tests are generally disliked because of the pain and inconvenience caused by finger pricking, resulting in fewer tests and inadequate blood glucose control. Poor blood glucose control results in more complications and even higher management costs. Particularly, repeated painful finger sticks are a major problem for young children and result in similar negative consequences for disease management.

Glucose sensing started in 1841 when it was performed in urine, but unfortunately the correlation between urine and plasma glucose was inconsistent [5]. Monitoring of blood glucose levels is currently the only recognized and widely used method for the diagnosis and control of diabetes. There are many different types of blood glucose meters on the market; however, they all require users to prick their fingers multiple times a day to obtain blood samples. Some minimally invasive or noninvasive techniques for blood glucose monitoring were studied, including infrared (IR) spectroscopy, fluorescence spectroscopy, Raman spectroscopy, and surface plasmon resonance. However, the results still have to be correlated with direct blood glucose measurements, and the sensitivity and reliability are limited by spectral signal-to-noise level and skin thickness. For example, in 2002, Cygnus Inc. introduced a wearable GlucWatch device measuring the glucose electroosmotically extracted across skin [6]. Nevertheless, the difficulty of use due to the sweat collection process and the low level of accuracy resulted in its removal from the market. Another product, the OrSense NBM device provided by OrSense Ltd. [7], which detects blood glucose concentration via an optical method called “occlusion spectroscopy” [8], has not achieved any significant success. Although optical technologies for glucose determination are available, most of them are for laboratory use due to the size, cost, and complexity of operation. Thus, a noninvasive, convenient, accurate, easy-to-use, portable, and low-cost diagnostic tool for diabetes is highly demanded.

As summarized by Lei et al. [9], there are three necessary prerequisites for most clinical applications: (i) a simple and inexpensive method for collecting biological samples with minimal discomfort, (ii) specific biomarkers associated with health or disease, and (iii) an accurate, portable and easy-to-use technology for disease diagnosis and health screening. Saliva, commonly considered as the ‘mirror of the body’, is very attractive as a bio-medium for clinical diagnostics. Its unique properties, such as noninvasive accessibility and the presence of plentiful disease biomarkers, make it particularly attractive for disease diagnosis and monitoring [10] and [11]. Saliva can be easily collected by individuals with modest instruction and it dramatically reduces the discomfort of the tests. Changes in saliva are believed to indicate the wellness of an individual. There are a large number of diagnostic analytes present in saliva, including glucose [12] and [13], steroid hormones [14], and the HIV antibody [15]. Saliva was first demonstrated to have diagnostic power comparable to that of blood in differentiating smokers from non-smokers through thiocyanate ions levels [16]. Results from blood, saliva, and urine as biomedia were compared and saliva was recognized as the most sensitive one. Saliva is also revealed to be more accurate than blood in detecting oral cancer [17] and [18]. Furthermore, the concentration of some other disease biomarkers in saliva was found to exceed that in blood, illustrating a further advantage of using saliva for clinical diagnostics [19] and [20].

Regarding to the technologies for determining salivary glucose levels, optical measuring systems such as Liquid Chromatography–Mass Spectrometry (LC–MS) and UV–VIS Spectrophotometry were reported [21] and [22]. However, the measurements can only be done in a laboratory as they require significant processing time, expensive reagents, sophisticated instrument, and highly trained professionals. Consequently, these methods cannot be used for individual glucose monitoring at home or in daily activities. Until now, there is not a suitable product for home care measurement of glucose using saliva. Technologies, including microchips and microfluidic devices, show great potential in developing a robust, cost-effective, accurate, portable, and easy-to-use diagnostic tool for saliva analysis [11] and [23]. Miniaturized saliva-based diagnostic technologies will enable the use of trace amount of biofluids to provide quick and reliable results for clinical decision-making and treatment outcomes-predicting.

A positive correlation between blood glucose and salivary glucose is revealed by many studies [21], [24], [25], [26] and [27]. Other than salivary glucose, no other parameters in saliva were found to be markedly affected in diabetes mellitus [13]. Therefore, salivary glucose can be utilized as an alternative diagnostic method for diabetes and as a general screen for prediabetes and undiagnosed diabetes.

Here we proposed an on-chip disposable nano-biosensor providing a painless test methodology with sufficient sensitivity. It is disposable and thus eliminates extensive cleaning or electrode pretreatment between measurements. The working electrode is functionalized with single-walled carbon nanotubes (SWNT) and multilayers of chitosan (CS), gold nanoparticles (GNp) and glucose oxidase (GOx), using a layer-by-layer (LBL) assembly technique [28]. The biosensor can detect glucose down to 0.1 mg/dL and provide noninvasive, reliable (high resolution), highly reproducible, convenient, fast, and continuous salivary glucose monitoring for personal and point-of-care use.

Results (English) 3:

全世界人民的生活与糖尿病和抽象
数百万数百万每年死于它。无创血糖检测方法，能够大大提高无痛的依从性和血糖控制，减少并发症和整体疾病管理成本。提供准确，成本低，和连续血糖监测，我们已经开发了一个独特的，一次性的唾液的纳米生物传感器。比实时无创唾液葡萄糖监测八个临床试验在两个健康个体进行（2–3小时期间每项试验，包括常规食品标准葡萄糖饮料的摄入量超过35个唾液样本）。优良的临床准确性发现相比，紫外分光光度法。通过测量受试者的唾液葡萄糖和并行血糖，我们发现两个生成的配置文件共享相同的变化趋势，但两者之间的关系是个人的依赖。有一个时间滞后的峰值血糖值之间的血液和唾液中的。然而，空腹血糖值之间的相关性是两个为每个人通过代替血液唾液使无创诊断糖尿病的常数。此外，良好的相关性唾液中的葡萄糖水平和血液中的前2小时后，观察到葡萄糖的摄取。血糖监测前和餐后2 h通常是由糖尿病患者对医生处方。因此，这种一次性生物传感器将实时唾液葡萄糖随时跟踪另一个。

关键词
无创；唾液；纳米生物传感器；一次性；血糖监测
1。介绍
国际糖尿病联合会估计全世界有382000000人患有糖尿病，2013，和数量预计将达到2035 592000000（增加55%）[ 1 ]。有5个。1000000型糖尿病相关的死亡在2013，相当于每6秒一人死亡，超过2011 [ 2 ] 11%增加。早期诊断，及时治疗，连续治疗是患者的生活质量至关重要，避免并发症如循环问题，肾功能衰竭，心脏病，中风，失明的[ 3 ]和[ 4 ]。糖尿病管理的现行做法，依靠监测血糖水平。血糖测量的要求来确定胰岛素用量、血糖检测异常指示的疾病，饮食结构的改变，或药物不良反应。这些侵入检测通常是因为厌恶的手指刺所造成的痛苦和不便，导致更少的测试不足和血糖控制。血糖控制不佳导致更多的并发症和管理的成本更高。特别是，重复的痛苦的手指是一个主要的问题儿童和类似的负面后果的疾病管理。

葡萄糖传感开始在1841时进行尿，但不幸的是，相关性尿和血浆葡萄糖不[ 5 ]。血糖监测是目前唯一的识别和控制糖尿病诊断中广泛使用的方法。有许多不同类型的市场上的血糖仪；然而，他们都需要用户刺破手指，一日多次获得的血液样本。研究了血糖监测一些微创或无创技术，包括红外（IR）光谱，荧光光谱，拉曼光谱，和表面等离子体共振。然而，结果还是要直接测量血糖的相关性，的灵敏度和可靠性是有限的光谱信号噪声水平和皮肤厚度。例如，在天鹅座2002，公司推出可穿戴glucwatch装置测量葡萄糖的电渗在皮肤中[ 6 ]。然而，由于难以用汗水收集过程和精度水平低导致其退出市场。另一种产品的公司，orsense [ 7 ]提供的orsense NBM装置，检测血糖浓度通过光学的方法称为“阻塞光谱”[ 8 ]，却没有取得任何重大成就。虽然葡萄糖测定的光学技术是可用的，其中大部分是实验室用由于尺寸，成本，和操作的复杂性。因此，一种非侵入性的，方便，准确，易于使用的，便携，廉价和糖尿病的诊断工具提出了更高的要求。

如雷等人总结。[ 9 ]，有临床应用三最必要的先决条件：（一）一个简单和廉价的具有最小的不适收集生物样本的方法，（ii）与健康或疾病相关的特异性生物标志物，及（iii）准确，便携式和易于使用的疾病诊断和健康筛选技术。唾液，普遍认为是“镜像的身体，是作为临床诊断的生物中非常有吸引力的。其独特的性能，如与大量的疾病的生物标志物的存在的无创性，使得它特别有吸引力的用于疾病的诊断和监测[ 10 ]和[ 11 ]。唾液可以用温和的指令的人容易收集，大大降低了测试的不适。变化在唾液被认为表示一个人的健康。有大量的诊断分析物存在于唾液，包括葡萄糖[ 12 ]和[ 13 ]，类固醇激素[ 14 ]，[ 15 ]和HIV抗体。唾液是第一个证明具有诊断能力相媲美的血在鉴别吸烟者非吸烟者通过硫氰酸根离子水平[ 16 ]。结果从血液，唾液，尿液和填料进行比较和唾液是公认的最敏感的人。唾液是还透露要比检测口腔癌[ 17 ]和[ 18 ]的血更准确。此外，唾液中的其他一些疾病的生物标志物的浓度被发现超过在血液，并进一步利用临床诊断[ 19 ]和[ 20 ]的唾液。

对于技术测定唾液中的葡萄糖水平，光学测量系统，如液相色谱–质谱（LC–MS）、紫外可见分光光度法测定–报道[ 21 ]和[ 22 ]。然而，测量只能在一个实验室，他们需要大量的处理时间，昂贵的试剂，精密仪器，和训练有素的专业人员。因此，这些方法不能用于个人的血糖监测在家里或在日常活动。直到现在，没有使用唾液葡萄糖的家庭护理测量一个合适的产品。技术，包括芯片和微流控设备，发展一个强大的，具有成本效益的，准确的，便携式显示出巨大的潜力，和唾液分析[ 11 ]和[ 23 ]的易于使用的诊断工具。小型化的唾液诊断技术将使体液微量的使用提供快速和可靠的结果为临床决策和治疗结果的预测。

血糖和唾液葡萄糖呈正相关关系是由许多研究发现[ 21 ]，[ 24 ]，[ 25 ]，[ 26 ]和[ 27 ]。除了唾液葡萄糖，唾液中没有其他的参数被认为是糖尿病[ 13 ]显著影响。因此，唾液葡萄糖可作为糖尿病的诊断方法和替代糖尿病前期和糖尿病一般屏幕。

这里我们提出了一个片上一次性的纳米生物传感器提供了一种无痛性的测试方法具有足够的灵敏度。它是一次性的，从而消除了测量之间广泛的清洁或电极预处理。工作电极与单壁碳纳米管的功能化（SWNT）和多层壳聚糖（CS），金纳米粒子（国民生产总值）和葡萄糖氧化酶（GOX），使用层由层（LBL）组装技术[ 28 ]。该传感器可以检测血糖下降到0。1毫克/分升，提供非侵入性的，可靠的（高分辨率），重现性好，方便，快速，为照顾个人点连续唾液葡萄糖监测。