Persistent Luminescence Nanophosphor Involved Near-Infrared

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Persistent Luminescence Nanophosphor Involved NearInfrared Optical Bioimaging for Investigation of Food-Borne Probiotics Bio-Distribution in vivo: A Proof-of-Concept Study Yao-Yao Liu, Jing-Min Liu, Dong-Dong Zhang, Kun Ge, Peihua Wang, Huilin Liu, Guozhen Fang, and Shuo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02870 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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Journal of Agricultural and Food Chemistry

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Persistent

Luminescence

Nanophosphor

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Bioimaging for Investigation of Food-Borne Probiotics Bio-Distribution in vivo:

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A Proof-of-Concept Study

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Yaoyao Liu†,∆, Jing-Min Liu§,∆, Dongdong Zhang†, Kun Ge†, Peihua Wang†, Huilin

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Liu ‡, Guozhen Fang*,†, and Shuo Wang*,§

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†

Near-Infrared

Optical

Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin, 300457, China.

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8

§

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University, Tianjin 300071, China

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Involved

‡

Research Center of Food Science and Human Health, School of Medicine, Nankai

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing, 100048, China.

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12

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* Corresponding authors

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(Guozhen Fang) Mail to: No 29, 13th Avenue, Tianjin Economic and Developmental

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Area (TEDA), Tianjin 300457, China.

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[email protected], Tel: +86-22-60912493; Fax: +86 22 6091 2493

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(Shuo Wang) Mail to: No.94 Weijin Road, Tianjin, 300071, China.

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[email protected]

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∆

These authors contributed equally to the work. 1 ACS Paragon Plus Environment


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ABSTRACT

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Probiotics has attracted great attention in food nutrition and safety research field, but

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thus far there are limited analytical techniques for visualized and real-time monitoring

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of the probiotics when it is ingested in vivo. Herein, the optical bioimaging technique

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has been introduced for investigation of food-borne probiotics bio-distribution in vivo,

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employing the near infrared (NIR) emitting persistent luminescence nanophosphors

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(PLNPs) of Cr3+-doped zinc gallogermanate (ZGGO) as the contrast nanoprobes. The

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ultra-brightness, super long afterglow, polydispersed size, low toxicity and excellent

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photostability and biocompatibility of PLNPs were demonstrated qualified as a tracer

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for labeling probiotics via the antibody (Anti-Gram positive bacteria LTA antibody)

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recognition as well as contrast agent for long-term bioimaging the probiotics. In vivo

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optical bioimaging assay showed that the LTA antibody functionalized ZGGO

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nanoprobes that could be efficiently tagged to the probiobics were successfully

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applied for real-time monitoring and non-damaged probing the bio-distribution of

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probiotics inside the living body after oral administration. This work presented a

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proof-of-concept that exploited the bioimaging methodology for real-time and

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non-damaged researching the food-borne probiotics behaviors in vivo, which would

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open up a novel way of food safety detection and nutrition investigation.

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KEYWORDS: Persistent luminescence, Probiotics, Antibody, Near infrared,

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Bioimaging

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INTRODUCTION

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A large number of bacteria in human intestine constitute one of the most complex

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ecosystems, providing a significant contribution to human biology and development1.

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In this microecosystem, the host and its microbiota develop a direct symbiotic and

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mutually beneficial relationship that affects the physical function of the host2, 3.

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Intestinal microbiota could enhance ferment the diet that the host unable to digest,

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synthesize low weight molecules to assist host nutrition, facilitate maturation of the

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epithelial barrier, influence intestinal immune development and enhance the host

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defense mechanisms4-7. In the past decades, the importance of the gut microbiota to

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human health and disease has been discussed a lot, which reach the conclusion that

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intestinal microflora not only contribute to creature innate and adaptive immune

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system, but also to the immune homeostasis and metabolic8.

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Probiotics bacteria, an important group of the gut microbiota, confer the major

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health benefit to the consumers when administered in adequate amounts9, 10. The

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action mechanisms were reported that probiotics bacteria could regulate and

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conducive to host healthy11, 12. Probiotics not only improve the competitive exclusion

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of the pathogenic bacteria and regulate the activity of the endogenous microorganisms,

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but also strengthen epithelial barrier function via immunoregulation signaling

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pattern13,

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Simultaneously, probiotics can motivate the immune system behavior of the host

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through transient predominance in the small intestine region that possesses the vast

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majority of the immune responses16, 17. In the patient with inflammatory bowel disease

14

or generate the immunomodulatory proteins to prevent apoptosis15.



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(IBD), it is found that the disease of IBD is closely related to microbial dysbiosis and

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the probiotics have ability to restore this imbalance to normal18. In addition to the

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innate factors, the acquired dietary pattern have a great influence of the intestinal flora

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colonization pattern, therefore, there is a proposal that offering the possibility of a

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new route for ingestion nutrition and intervention therapy, such as orally up-taking

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food containing live bacteria (such as yoghurt, cultured buttermilk, fermented

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vegetable juice), of which the probiotics are supposed to partly colonize in

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gastrointestinal tract to exert function19.

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In spite of the well-studied benefits and special functions to human health, the

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metabolism, distribution and related immunomodulation of probiotics in vivo is still

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currently poorly understood and the function knowledge and general action of most

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new species are remain unidentified and need to be assessed to acquire more

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information. With advances of the modern molecular-based biotechnology, various

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methods, such as polymerase chain reaction combined with denaturing gradient gel

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electrophoresis (PCR-DGGE), real-time PCR, DNA microarray, pyrosequencing, and

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high-throughput sequencing, have been proposed to determine the DNA sequences of

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biological samples to reveal the types, distribution, and metabolism of probiotics and

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their relative abundance in gut20-22. However, the above mentioned techniques usually

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need to harvest the samples (faeces or vivo tissue), design probes/primers, and

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time-consuming statistical analysis. Moreover, these methods do not allow in situ

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detection, which can hardly provide the real scene of probiotics behaviors inside the

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living body with non-destructive and real-time in situ monitoring. Karimi S et al have 4 ACS Paragon Plus Environment

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constructed the plasmids carrying reporter genes expressing fluorescent and

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luminescent proteins, then cloned it into two L. reuteri strains, resulting in the bacteria

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emitting bright signals of fluorescence and luminescence to reveal the localization and

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distribution of the probiotics in the gut by in vivo and in vitro imaging23. Fluorescein

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isothiocyanate (FITC) has been applied to label probiotics by Xing et al, combined

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with fluorescence imaging to investigate modulation of gut microbiota and the

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distribution and colonization of L. kefiranofaciens in the mouse intestinal tract24.

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In vivo optical bioimaging with the ability of non-invasive conceive of structural

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and functional processes at the cellular and molecular level, appears as the ideal

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methodology for investigation of probiotics behaviors after uptaken in the living

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body25. Bioimaging has become the indispensable tool for establishing a means of

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real-time monitoring and non-damaged detection within the living body for diagnosis

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and therapy in biomedical fields

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environment in vivo

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bio-labels, and the current research focused on the development of highly efficient

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luminescent materials as fluorescence imaging probes, such as quantum dots (QDs)31,

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25-28

and monitoring the change of biological

29, 30

. The fluorescence imaging basically relied on luminescence

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, organic dyes33-35, fluorescent proteins36, 37, noble metal nanomaterials38-40, carbon

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dots41,

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, and upconversion nanoparticles43. However, most of them still have

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shortages of high photo-bleaching rate, poor signal-to-noise ratio, short luminescence

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lifetimes and poor biocompatibility25. Persistent luminescence nanophosphors (PLNPs)

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possess the remarkable optical phenomenon of the continuous afterglow in the

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near-infrared (NIR) spectral regions (650~1000 nm) for minutes, hours or even days 5 ACS Paragon Plus Environment


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after stoppage of the excitation that makes them qualified as a new generation of in

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vivo optical bioimaging nanoprobe44-47. The typical PLNPs, zinc gallogermanate

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(ZGGO) and zinc gallate (ZGO), have demonstrated several distinctive features: i) the

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ability to store the excitation energy and lentamente release by a photonic emission

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after removal of the excitation source; ii) no need of external excitation that leads to

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complete overcome of the tissue auto-fluorescence and significant enhancement of

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signal-to-noise ratio and sensitivity; iii) the adjustable red-NIR luminescence located

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in the region of tissue transparency window that further increases the detection depth;

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iv) the red-light re-excitable luminescence that favored the in vivo long-term

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bioimaging25, 48, 49. Therefore, in past few years, near-infrared persistent luminescence

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nanoparticles continuously arouse the extensive exploration interest of making them

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as biological imaging probes50-54.

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Herein, with the proof-of-concept, the optical bioimaging technique has been

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introduced for investigation of food-borne probiotics bio-distribution in vivo,

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employing the Cr3+-doped ZGGO PLNPs as the contrast nanoprobes. The PLNPs

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prepared via hydrothermal method combination with solid phase calcination in the air

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have demonstrated remarkable advantages, including ultra-brightness, super long

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afterglow, polydispersed size, low toxicity and excellent photostability and

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biocompatibility, which ensured the success of high-sensitivity and long-term

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bioimaging in the living systems. For the first time, PLNPs were utilized as targeted

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optical probes for selective tagging the food probiotics (Lactobacillus as the

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representative) with the assistant of Anti-Gram positive bacteria LTA antibody 6 ACS Paragon Plus Environment

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bioconjugated on the PLNPs surface. After oral administration, we can trace labeled

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bacteria to supervise their progress though the gastrointestinal tract in vivo after

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excitation, and acquaint their bio-distribution by animal optical detection.

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Consequently, this work has proposed a new perspective for in vivo investigation of

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food probiotics bio-distribution with the advantageous real-time monitoring and

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non-damage detection ability, and broadened the methodology development for food

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safety detection and nutrition investigation based on the advanced functional

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nanomaterials.

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MATERIALS AND METHODS

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Materials and Instrument. All reagents were of the highest available purity and at

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least of analytical grade. Ultrapure water (Milli-Q quality water system, 18.2MΩ cm,

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Millipore, USA) was used throughout all experiments. Zn(NO3)2•6H2O (99.99%),

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Ga2O3

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1-ethyl-3-(3-dimethylaminopropyl)

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N-hydroxysuccinimide (NHS), (3-aminopropyl) triethoxysilane (APTES) and

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dimethylformamide (DMF) were all purchased from Aladdin (Shanghai, China).

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Tert-butylamine, ammonium hydroxide, concentrated nitric acid and hydrochloride,

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NaCl, KCl, MgCl2, CaCl2, FeCl2, AgNO3, Cu(NO3)2, Zn(NO3)2, Al(NO3)3 and amino

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acids (Cys, Phe, Ala, Gly, Glu, Gln, Met, Arg, Lys, Tyr, Leu, Pro, Trp, Ser, Thr, Asp,

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Asn, Val, Ile, His), de Man, Rogosa and Sharpe culture medium (MRS) were all

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purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China), bovine

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serum albumin (BSA), bovine hemoglobin (BHB), glutathione (GSH), ovalbumin

(99.999%),

GeO2(99.999%),

Cr(NO3)3•6H2O

carbodiimide

hydrochloride


(99.99%), (EDC),


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(OVA), peroxidase (POD), Casein, phosphoeptide, thrombin, lysozyme, pepsase,

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pancreatin, cytochrome C (cyt-C) were purchased from Sigma-Aldrich (St Louis, MO,

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USA). Gram-positive Bacteria monoclonal antibody (BDI380) was obtained from

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Amyjet Scientific Inc (Wuhan, China). All glassware used in the experiment was

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soaked in a bath of freshly prepared aqua regia (HCl: HNO3= 3:1, v/v) and douched

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thoroughly with H2O before use (caution: aqua regia is dangerous and should be

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handled with care).

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The photoluminescence excitation, emission spectra and afterglow decay curves

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of ZGO and ZGGO were measured by a Lumina spectrofluorometer (Thermo Fisher

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Scientific, Waltham, MA, USA) equipped with continuous (150 W) and pulsed Xenon

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lamps as an excitation source and the phosphorescence signals were recorded using a

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standard

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microstructure, size and morphology of the prepared nanoparticles were observed and

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recorded by high resolution transmission electron microscopy (HRTEM) using on a

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JEM-2100F field emission transmission electron microscope (JEOL, Japan) operated

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at a 200 kV accelerating voltage. The biological section sample TEM images were

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obtained on HT7700 Hitachi (Japan) operating at a 100 kV accelerating voltage.

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X-ray diffraction (XRD) patterns was carried out by using a D/max-2500

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diffractometer (Rigaku, Japan) equipped with Cu Kα radiation (λ = 1.5418 Å).The

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elemental analysis was performed by inductively coupled plasma mass spectrometry

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(ICP-MS, Agilent 7500CX). The UV-vis absorption spectra were recorded on a

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UV-3600 UV-vis-NIR spectrophotometer (Shimadzu, Japan) with 1 cm path-length.

photo-multiplier

tube

(Hamamatsu,

R928) detectors.


The

crystal

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All the measurements were executed at room temperature. Fourier transform infrared

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(FT-IR) spectra (4000-400 cm-1) in KBr were recorded on a Magna-560 spectrometer

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(Nicolet, Madison, WI).

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Synthesis

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Zn1.25Ga1.5Ge0.25O4:0.5%Cr3+ nanoparticles were both synthesized by hydrothermal

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method combination with calcinations in air referring to the reported procedures55, 56.

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ZGO PLNPs: 0.013 mmol chromium nitrate and 3.01 mmol zinc nitrate dissolved in

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10 mL ultrapure water were mixed with 10 mL of gallium nitrate (0.6 M) solution

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under vigorous stirring. Subsequently, the white precipitated precursor was obtained

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by adding the tert-butylamine to adjust the pH to 7.5. After a 3-h of stirring and

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ultrasonic treatment at room temperature, the reaction solution was transferred into a

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50-mL teflon-lined stainless steel autoclave and treated under 120 °C for 24 h. After

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cooled to room temperature, the resulting compound was washed three times with

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water and ethanol, respectively, and finally annealed in air at 750 °C for 5 h. The

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obtained PLNPs powder was grinded after a freeze-drying process, and stored in dark.

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ZGGO PLNPs: The synthesis procedure of Zn1.25Ga1.5Ge0.25O4:0.5%Cr3+ was similar

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to that of ZGO with some modifications. In brief, zinc nitrate (5 mmol), chromium

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nitrate (0.3 mmol), and 10 mL of ammonium germinate (0.1 M) was mixed with 10

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mL of gallium nitrate (0.6 M) aqueous solution under vigorous stirring.

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tert-butylamine was rapidly added to adjust the pH to 8, and the turbid liquid was kept

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stirring for 1 h followed by an ultrasonic treatment at room temperature. The obtained

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mixture was then transferred into a 50-mL Teflon-lined stainless steel autoclave for a 9

of ZGO

and ZGGO

PLNPs.

The ZnGa1.995O4:0.5%Cr3+ and

ACS Paragon Plus Environment

The


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15-h heat treatment at 120 °C. The resulting precipitate was washed three times with

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water and ethanol, respectively, and finally annealed in air at 1000 °C for 1 h. The

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precipitate powder was ground with a mortar and pestle, and stored in dark after

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freeze-drying.

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Surface functionalization of PLNPs. The surface amino functionalization of PLNPs

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was performed via the Stöber procedure with APTES as silane coupling agent

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according to the previous literature44, 57. The ZGGO powder was wet ground with

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alcohol for three times. Subsequently, the sample was suspended in the NaOH (5 mM)

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solution under ultrasonic treatment for 1 h, and vigorously stirred overnight at room

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temperature. The white colloidal suspension solution was first selected by

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centrifugation at 4500 rpm for 10 min to remove the large sized particles. Then the

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supernatants were gathered and centrifuged at 3500 rpm for 15 min to collect the

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nanoparticles with 20~50 nm diameter. The obtained freeze-drying precipitate (5 mg)

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was resuspended in 2 mL DMF by sonication, followed by dropwise addition of 20 µL

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APTES to initiate the formation of the NH2-ZGGO. The reaction was kept under

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vigorous stirring at 80 °C for 24 h. Finally, the resulted NH2-ZGGO nanoparticles

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were washed with DMF to remove the unreacted APTES and gathered by

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centrifugation.

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Preparation of antibody-ZGGO conjugates. Anti-Gram positive bacteria LTA

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[3801] monoclonal antibody was immobilized on ZGGO nanoparticles surface

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according to the literature method with some modification58, 59. Typically, 1 mg of

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antibody was dissolved in 10 mL PBS (0.1 M, pH 7.4), followed by addition of NHS 10 ACS Paragon Plus Environment

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(15 mg) and EDC (15 mg). The mixture was incubated for 30 min at room

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temperature to fully activate the carboxyl group of the antibody. NH2-ZGGO

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suspension solution was obtained by adding 10 mg of NH2-ZGGO power into 10 mL

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PBS (0.1 M, pH 7.4) buffer and supersonic treatment for 30 min. The above

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mentioned two kinds of solution were mixed together thoroughly for another 4 h

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reaction at room temperature under gently stirred. The resulting product

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antibody-ZGGO was centrifuged (4 °C, 7000 rpm, 5 min) and washed with PBS

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buffer for three times to obtain the conjugation of antibody-ZGG, freeze drying, and

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stored at 4 °C for standby.

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Strain and microbial cultures conditions. A bacterial strain ZW-128 of lactobacillus

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reuteri [“Gram-positive group”] in this study is available from the animal resources

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development and functional food Lab at the College of Food engineering and

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biotechnology, Tianjin university of science and technology, Tianjin, China, which

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was used as target and bioimaging in all experiments. Pure lactobacillus reuteri

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cultures were prepared by activating twice from -80 °C frozen inventories in de Man,

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Rogosa and Sharpe (MRS) broth at 37 °C under shake-flask culturing for 12 h to

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achieve logarithmic growth phases. In experiments, 3 mL of the bacterial culture was

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added to 100 mL of MRS broth medium and incubated overnight at 37 °C. Cell pellets

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was harvested by centrifugation (6000 × g for 10 min) at 4 °C from the overnight

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grown cultural suspension, the supernatant was removed and the pellet subsequently

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washed twice with sterilized PBS (0.01 M, pH 7.4) buffer and adjusted the bacterial

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concentrations by determining the optical density at 600 nm (OD600). 11 ACS Paragon Plus Environment


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Preparation of L. reuteri competent cell. The competent cell was prepared

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according to the previously method with some modification60. 25 mL of preculture L.

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reuteri cell in logarithmic growth phase at 37 °C was inoculated into 100 mL

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sterilized MRS medium supplemented with 1% (w/v) glycine. The culture was

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incubated at 37°C until the cell was at exponential phase (OD600 = 0.6), then the cells

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were cooled on an ice-bath for 10 min and harvested by centrifugation at 4 °C, 3000 ×

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g for 5 min and remove the supernatant softly (the parameters depend on vary strains).

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The pellet of cells was washed with 100 mL of pre-cooling 10 mM MgCl2 and

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centrifuged at 4 °C, 3000 × g for 5 min. Afterwards, cells were resuspended charily in

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100 mL of pre-cold SM solution (925 mM sucrose and 3.5 mM MgCl2), and the pellet

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of thallus was collected by centrifugation (5000 × g, 10 min) at 4 °C. After repeating

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this step twice, cells were resuspended gently in 1 mL of pre-cooling SM solution and

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stored on ice-bath before further treatment, of which process should be implemented

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as quickly as possible.

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Interaction of ZGGO with L. reuteri. Three different incubation ways between L.

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reuteri cell suspension (109 cfu mL-1) and ZGGO nanoparticles have been compared. i)

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Competent cells (60 µL) prepared as described above were mixed with 40 µL of

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ZGGO nanoparticles dispersion in PBS (1 mg mL-1) and suffered from an electric

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pulse in a 0.1 cm cuvette by using a Gene Pulser and a Pulse Controller apparatus

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(Gene Pulser Xcell, Bio-Rad, USA). Immediately, the cells was gained by

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centrifuging the above mixture at 4 °C, 1500 × g for 5 min, and decanted the

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supernatant. ii) The competent cells (1 mL) were treated with ZGGO nanoparticles 12 ACS Paragon Plus Environment

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(500 µL, 1 mg mL-1) under softly shaking at 37 °C for 6 h and washed with PBS twice

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(0.01 M, pH 7.4). iii) Meanwhile, bacteria (L. reuteri) from logarithmic phase were

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obtained by rinsing with PBS (0.01 M, pH 7.4) twice and resuspending them in PBS,

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which was mixed with antibody-ZGGO nanoparticles and incubated for 1 h at 37 °C.

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As the control, suspended bacterial cell in PBS without any treatment was interacted

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directly with ZGGO nanoparticles under softly shaking at 37 °C for 6 h. After treating,

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all above bacterial cell was fixed with 2.5% glutaraldehyde in PBS solution right

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away to the pellet in the tube and stored at 4 °C overnight for TEM samples

268

preparation.

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The interaction of ZGGO with L. reuteri was evaluated via TEM. In a typical

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assay, the slice sample was prepared as follows: The above-mentioned pellet was

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washed with PBS for three times to remove the glutaraldehyde, then incubated with

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2% osmium tetraoxide (OsO4) in PBS at 4°C for 30 min. The superfluous OsO4 was

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eliminated by washing with PBS, and the fixed pellet was dehydrated through

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different levels of ethanol (30%, 50%, 70%, 90%), respectively, followed by series

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wash using 90% ethanol and 90% acetone (1:1, v/v), 90% acetone, and 100% acetone

276

(3 times) for 20 min at the room temperature. The pellet sample was prepared by

277

immersing into araldite resin medium overnight and embedding into the resin. The

278

sample was cured at 60 °C overnight in a vacuum oven until it thoroughly infiltrated

279

in the resin. Using an ultra-microtome (Power Tome-PC, RMC, USA) cut the

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resin-embedded pellet into 60~70 nm ultrathin slices which were then put on the Cu

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grids for TEM examination. 13 ACS Paragon Plus Environment


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Evaluation of ZGO and ZGGO stability. 20 mg of prepared ZGO and ZGGO

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nanoparticles were infiltrated into 20 mL of the PBS, HEPES, HBSS, Tris-HCl,

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artificial gastric juice, artificial intestinal juice, and ultrapure water, respectively. The

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element content of Zn, Ga and Cr in the supernate of materials suspension was

286

monitored by ICP-MS analysis at the time of the 0, 1, 2, 3, 4, 5, 6,12, 24, 48 h. In

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addition, prepared nanoprobes solid solutions were separately incubated with 9

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species of metal salts, 20 kinds of amino acids, 12 common biomolecules and

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ultrapure water to record the change of phosphorescence intensity (excitation 295 nm,

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emission 698 nm) to evaluate the photostability and biocompatibility of ZGO and

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ZGGO.

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Evaluation of the ZGGO toxicity. The methyl thiazolyl tetrazolium (MTT) assay

293

were performed to determine the in vitro cytotoxicity of the ZGGO nanoprobes.

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Mouse embryo fibroblast cell lines (Balb/3T3), human cervical carcinoma cell lines

295

(Hela), and human breast carcinoma cell lines (MCF-7) were obtained from China

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Center for Type Culture Collection (Wuhan, China). Briefly, the three cell lines were

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plated at a density of 4×104 cells per well in 96-well plates and grown for 24 h at

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37°C in 5% CO2. The NH2-ZGGO nanoparticles dispersed in 10 mM PBS solution

299

with a wide concentration range from 50 to 1000 µg mL-1 were subsequently added

300

into the cell and incubated for another 24 h under the same conditions as above. MTT

301

(10 µL, 3 mg mL-1) was added to each well, and incubated for another 4 h at 37 oC.

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Then, 150 µL DMSO was added to each well, and the plate was stayed at room

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temperature for 10 min. OD570 (Abs. value) of each well was measured by the 14 ACS Paragon Plus Environment

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Multiskan Spectrum multifunction microplate reader (Labsystems, Thermo, USA).

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In vivo luminescence imaging. The adult athymic BALB/c mice (15~20 g) were

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obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All animal

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experiments were implemented in accordance with guidelines of Tianjin Committee

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of Use and Care of Laboratory Animals, and all project protocols were approved by

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the Animal Ethics Committee of Nankai University. In vivo experiments were

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performed on anesthetized mice with chloral hydrate (200 µL, 4%). The prepared L.

311

reuteri-ZGGO (2 mL, 1×109 cfu mL-1) or ZGGO (2 mL, 1 mg mL-1) dispersed in 10

312

mM sterile PBS solution was given to the mice through oral administration,

313

respectively. The nude mouse was fasted for 36 h before oral administration of

314

materials. After 120 min of gavage, the mice were excited with a red LED light (650 ±

315

10 nm) for 60 s to reactivate the persistent luminescence of ZGGO and the signal

316

acquisition was resumed under the optima camera.

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RESULTS AND DISCUSSION

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Principle of in vivo investigation of probiotics bio-distribution. As illustrated in

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Figure 1, in order to directly track the bio-distribution of probiotics inside the living

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body, lactic acid bacillus was labeled by antibody-ZGGO nanoprobe with ultra-bright

321

long afterglow luminescence and the bioimaging assay was performed post oral

322

administration. First of all, the surface of PLNPs was activated with considerable

323

amount of hydroxyl groups through 5 mM NaOH treatment (OH-PLNPs).

324

Subsequently, amination of the PLNPs was formed by Stöber method via the reaction

325

of the silanol groups of APTES with the surface hydroxyl groups of PLNPs, making 15 ACS Paragon Plus Environment


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amino group decorated onto the ZGGO PLNPs surface (NH2-PLNPs). Afterwards,

327

with the aid of NHS-EDC catalysis, the LTA antibody could react with the -NH2

328

groups on the surface of NH2-PLNPs via carbodiimide method to obtain binding sites

329

for the probiotics and then produced the antibody-PLNPs nanoprobes, which will be

330

used to combine with the lipoteichoic acid (LTA) antigen on surface of probiotics.

331

Finally, the antibody-PLNPs nanoprobes were employed as luminescence tagging for

332

in vivo bioimaging of the labelled probiotics after oral administration.

333

Preparation and characterization of the ZGO and ZGGO PLNPs. The Cr3+-doped

334

ZGO and ZGGO persistent luminescence nanoparticles were successfully synthesized

335

in the form of white powder by hydrothermal method combined with calcination in air,

336

according to the reported methods with slight modifications55, 56. In the ZGO and

337

ZGGO PLNPs nanostructure, zinc gallate acts as the host, while the Cr3+ and Ge4+ are

338

the doped ions, as the luminescent centers and defect centers (vacancies), respectively.

339

Depending on the crystal-field environment of the host lattices, Cr3+ ions act as a

340

favorable emitter, which have a broadband NIR emission from 650 to 950 nm,

341

overlapping with the biologically transparent window (650-1100 nm)61, 62. As the

342

defect center, Ge4+ is responsible to extend the afterglow time in the persistent

343

luminescence nanostructures. Figure 2 shows the normalized excitation, emission

344

spectra and NIR persistent luminescence of the ZGO and ZGGO powder at the room

345

temperature. After excited at 260 nm, the prepared ZGO and ZGGO nanophosphors

346

both showed NIR emissions centered at 694 nm with a broad emission band from 650

347

nm to 900 nm. The excitation spectra of ZGO and ZGGO monitored at 694 nm is 16 ACS Paragon Plus Environment

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corresponding of three excitation broad bands due to the inner transitions of Cr3+,

349

which possessed a broad excitation range from 200 nm to 600 nm.

350

In addition to the intense and broad NIR photoluminescence, the as-prepared

351

ZGO and ZGGO both demonstrated remarkable long afterglow features, with the

352

persistent luminescence lifetime over 90 h and 150 h, respectively, which formed the

353

basis of long-term bioimaging application. (Figure 3D and 3H) Through the

354

comparison of their afterglow decay curves, ZGGO showed better afterglow

355

properties than ZGO due to the presence of Ge4+ ion in the lattices. The nanocrystal

356

phase was confirmed by XRD analysis, which indicated the XRD pattern of ZGO and

357

ZGGO were consistent with the spinel phase of ZnGa2O4 (JCPDS no. 38-1240) and

358

Zn2GeO4 (JCPDS no. 25-1018), respectively. (Figure 2C)

359

HRTEM characterization revealed that both ZGO and ZGGO PLNPs were

360

generated with the diameter ranging from 30 to 80 nm and good dispersity. Compared

361

with the traditional high temperature solid calcination method, the hydrothermal

362

method could effectively control the nanoparticle size and achieve improved

363

dispersity of PLNPs. Hydrodynamic diameter of the nanoparticles measured by

364

dynamic light scattering was 45 ± 5 nm (ZGGO) and 48 ± 6 nm (ZGO),

365

respectively.(Figure 3 C and 3G) The elemental composition of ZGO and ZGGO

366

PLNPs was further confirmed by the EDX analysis, which showed the presence of Ge

367

elements in the ZGGO nanostructure.( Figure 3 B and 3F)

368

Biocompatibility, stability and toxicity of PLNPs. Before application of the

369

synthesized PLNPs for in vivo bioimaging, the biocompatibility, stability and toxicity 17 ACS Paragon Plus Environment


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of the nanoprobes have been integrally evaluated. The biocompatibility of PLNPs was

371

evaluated via determining the photoluminescence intensity changes of ZGO and

372

ZGGO nanomaterials in the presence of various substances inside the living body,

373

including metal ions, amino acids, and proteins. As shown in Figure 4, the tested

374

metal ions, common amino acids, proteins and enzymes produced negligible effect to

375

the luminescence of PLNPs nanomaterials (ZGO and ZGGO), which proved that

376

ZGGO and ZGO PLNPs both possessed excellent biocompatibility in vivo. For the

377

investigation of the chemical stability of PLNPs, the ZGO and ZGGO powders were

378

separately incubated with different media, including ultrapure water, PBS (10 mM,

379

pH 7.4), Tris-HCl buffer (10 mM, pH 7.4), HEPES buffer (10 mM, pH 7.5), HBSS

380

buffer,

381

contents of the supernates of the solid solutions were measured by ICP-MS elemental

382

analysis. After a 48-h continuous monitoring, the results of the metal element contents

383

in the supernatant revealed there was few Ga3+, Zn2+ and Cr3+ ions leakage from the

384

nanocrystals when treated by the seven media. Therefore, it was proved that the

385

PLNPs nanoparticles can maintain long-time structure stability existing in various

386

biological fluids, which significantly favored the following in vivo bioimaging assay

387

via oral administration. In consideration of the better afterglow performance and

388

comparable biocompatibility and chemical stability, ZGGO PLNPs were finally

389

chosen as the optical centers for the following biolabeling and bioimaging assays.

simulated intestinal fluid, and simulated gastric juice. Then the element

390

The in vitro cytotoxicity of the ZGGO nanoprobes was assessed via cell counting

391

assay performing on three types of cell lines, 3T3 normal cell, MCF-7 cancer cell and 18 ACS Paragon Plus Environment

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Hela cancer cell. As shown in Figure 6A, after exposure to the concentration as high

393

as 1000 µg mL-1 of ZGGO nanoprobes for 24 h, the viability of three type cells was

394

still higher than 80%, which proved no significant toxicity of the ZGGO PLNPs to

395

three type cells. Furthermore, ZGGO PLNPs was fed to mice and the body weight

396

change of the mice was continuously recorded to evaluate the long-term in vivo

397

toxicity. After 30 days of ZGGO PLNPs oral administration, the mice viability was

398

still 100%, and there was no significant difference of the body weights between

399

control and treated groups (Figure 6B). Based on above phenomenon, the ZGGO

400

PLNPs were proved to be low toxic and suitable for in vivo imaging.

401

Surface functionalization of ZGGO PLNPs. To reinforce the targeted specificity,

402

Anti-Gram positive bacteria LTA antibody was grafted onto the surface of the ZGGO

403

PLNPs via the NHS-EDC assisted amidation reaction. The surface functionalization

404

with APTES and antibody to ZGGO PLNPs were confirmed by FT-IR analysis

405

(Figure 6C) and Zeta potential (Figure 6E). FT-IR spectra showed there appeared

406

strong absorption band at 3437 cm-1 (stretching vibrations of O-H), manifesting the

407

successful modification of hydroxyl groups. The strong FT-IR band at 1011 and 1115

408

cm-1 (stretching vibrations of O-Si-O), 2938 cm-1 (-CH2 stretching bands) and 3421

409

cm-1 (N-H stretching bands) indicated the successful modification with APTES. The

410

FT-IR absorption bands of antibody-ZGGO at 1716 cm-1 (stretching vibration of C=O)

411

suggested the antibody successfully grafted onto the surface of the NH2-ZGGO. With

412

the different surface modification process, the surface zeta potentials change of the

413

ZGGO nanoparticles were used to verify the state of materials surface functional 19 ACS Paragon Plus Environment


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group. After treatment with NaOH, the ZGGO revealed negative zeta potential of

415

-30.9 mV due to the presence of hydroxyl groups on surface. Then the zeta potential

416

changed to +12.9 mV after the APTES silane coupling reaction, proving the

417

decoration of –NH2 on particle surface. Subsequently, binding of antibody with the

418

NH2-ZGGO produced negatively-charged surface of -14.4 mV. All the above evidence

419

conformed that the successful preparation of the bioconjugate of antibody-ZGGO

420

nanoprobes. In addition, the luminescence intensity of NH2-PLNPs maintained over

421

80% after soaking in ultrapure water for 30 days, indicating its excellent

422

photostability. (Figure 6F) Meanwhile, there was not much loss of luminescence

423

intensity of PLNPs after modified with antibody or binding with L. reuteri (Figure

424

6D).

425

Interaction of PLNPs nanoprobes with probiotics. In order to realize the optimal

426

labeling of antibody-ZGGO nanoprobes with to the targeted Lactobacillus, two

427

interaction approaches have been explored, internalization and antibody-antigen

428

specific binding.

429

It is well-studied that nanoparticles can be internalized via receptor-mediated

430

endocytosis by some special mammalian cells, such as macrophages, hemameba and

431

neutrophils63, 64. However, it is generally agreed that bacterial cells hardly support

432

endocytosis, pinocytosis or exocytosis due to the presence of the thick peptidoglycan

433

cell wall65, 66. TEM analysis of sample thin sections can be able to directly visualize

434

the states of the microorganisms upon incubation with ZGGO (1 mg mL-1) in

435

sterilized PBS (Figure 7). The L. reuteri Gram-positive cell with the typical tubular 20 ACS Paragon Plus Environment

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shape demonstrated complete cell-structure and membrane-layer. After incubation,

437

most of the nanoparticles existed away from the cells, and failed to label to the cells

438

of interest owing to the thick cell wall of bacteria. Therefore, electroporation

439

treatment was carried out to facilitate the bacterial cells to uptake the nanoparticles.

440

Primitively, the competent L. reuteri cells with better penetrability than the normal

441

cells were homogeneously mixed with ZGGO nanoprobes, followed by the

442

electroporation treatment immediately, and incubated at 37 oC for 6 h. The same

443

process was performed on the competent cells without the following electroporation

444

treatment as a control. However, similar results were obtained that limited amount of

445

nanoparticles can penetrate the cell membrane into the cells, of which amount was

446

insufficient to realize the luminescence tagging for the following bioimaging assay

447

(Figure 8A-D).

448

In the following assay, the specific immunoreaction was introduced for

449

bio-labeling probiotics. The antibody against the LTA antigen on the surface of L.

450

reuteri Gram-positive cells was modified to the NH2-ZGGO nanoparticles to make the

451

antibody-ZGGO nanoprobes that can interact with L. reuteri Gram-positive cells via

452

surface antigen-antibody binding. TEM results strongly support the idea that

453

considerable amount of nanoparticles were decorated onto the bacteria surface via the

454

immunoreaction of antibody with the LTA antigen epitopes on the cell wall of the L.

455

reuteri bacteria, leading to the nanoparticles embraced around the cells as clearly

456

shown in Figure 8E-F. Consequently, the ZGGO PLNPs have been successfully

457

labeled to the targeted probiotics via the immunoreaction to form the L. 21 ACS Paragon Plus Environment


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reuteri-ZGGO bioconjugates for the following bioimaging application. In order to

459

assess the activity of Lactobacillus cells after labeling, the bacterial activity also has

460

been investigated after modification of the antibody. The activity of the bacterial after

461

labeling and untreated bacterial were measured by Bacterial activity kit-8 (CCK-8)

462

assay (Bestbio Biotech, Shanghai, China), respectively. After 12 h or 24 h treatment,

463

the viability of the activity of the bacterial were still higher than 80%, which proved

464

there is no significant influence of nanoprobes to the bacterial activity. (Figure 9)

465

To understand the adhesion performance of probiotics after labeling with

466

nanoprobes, the MC38 cells were used to make a simple contrast test to investigate

467

the changes of the adhesion performance of probiotics before and after modification

468

through in vitro culture by the method of counting viable bacteria67. The results

469

showed that the adhesion performance of the ZGGO- Lactobacillus has declined 34%

470

compared with the L. reuteri without modification. This indicates that although parts

471

of the LTA on the surface of bacteria are occupied by antibody-ZGGO conjugates, the

472

bacteria maintained significant adhesion ability to the surface of cells in gut. (MC38

473

cells were purchased from National Infrastructure of Cell Line Resource, China and

474

cultured in RPMI Medium 1640 basic with 10% fetal bovine serum. Cells were

475

cultured in the 37 ℃ incubator contained 5% CO2.) The above results supported our

476

principle of nano-imaging guided in vivo investigation study of bacteria.

477

Bio-distribution and orally administrated in vivo bioimaging. In order to

478

intuitively monitor the bio-distribution of the probiotics L. reuteri inside the living

479

body, the prepared L. reuteri-ZGGO was orally administrated into mouse by gavage. 22 ACS Paragon Plus Environment

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As shown in Figure 10B, NIR light emitting persistent luminescence of L.

481

reuteri-ZGGO appeared in the stomach 1 min after oral administration, subsequently

482

arrived at intestinal tract and spread around the whole digestive tract region of mouse

483

6 min later, and became obvious in the intestine region at 30 min. After 60 min, L.

484

reuteri-ZGGO still remained in the stomach and intestines site, probably due to the

485

gradually adhesion of L. reuteri cells in the gastrointestinal tract. Although the

486

persistent luminescence intensity of ZGGO gradually decreased without in situ

487

excitation, the signal to noise ratio was still >20 at 120 min post oral administration.

488

NH2-ZGGO nanoparticles were orally administrated via the same procedure as a

489

control. The persistent luminescence appeared in intestinal tract, spread around the

490

whole body within 6 min, and gradually disappeared 120 min post oral administration,

491

without noteworthy accumulation in the gastrointestinal tract.

492

After 120 min oral administration, the mice were re-irradiated by a LED light to

493

re-activate ZGGO (Figure 10). It was observed that NH2-ZGGO nanoparticles in

494

control group spread over the whole mice body and mainly accumulated in the liver

495

and spleen, whereas the bio-distribution of the L. reuteri-ZGGO remained in the

496

digestive tract areas. Furthermore, the in vivo distribution of antibody-ZGGO labeled

497

Lactobacillus and NH2-ZGGO has been further evaluated by anatomy experiment

498

followed by fluorescence and ICP-MS measurements of organs, of which results were

499

consistent with those of bioimaging assay. (Figure 10C and 10D) The above results

500

demonstrated the L. reuteri-ZGGO bioconjugates could effectively tract the

501

bio-distribution of probiotics inside the living body, and as a proof-of-concept, the 23 ACS Paragon Plus Environment


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PLNPs-based long-term optical bioimaging was able to be utilized for probing the

503

bacteria behaviors in vivo.

504

In summary, the optical bioimaging technique has been introduced for

505

investigation of food-borne probiotics bio-distribution in vivo, employing the ZGGO

506

PLNPs as the contrast nanoprobes. The ultra-brightness, super long afterglow,

507

polydispersed size, low toxicity and excellent photostability and biocompatibility of

508

PLNPs were demonstrated qualified as a tracer for labeling probiotics via the antibody

509

recognition as well as contrast agent for long-term bioimaging the probiotics. This

510

work has proposed a new perspective for in vivo investigation of food probiotics

511

bio-distribution with the advantageous real-time monitoring and non-damage

512

detection ability, and broadened the methodology development for food safety

513

detection and nutrition investigation based on the NIR PLNPs assisted in vivo

514

bioimaging assay.

515

ACKNOWLEDGMENTS

516

This study was funded by the Ministry of Science and Technology of China (No.

517

2012AA101602). This work was supported by International Science and Technology

518

Cooperation Program of China (No.2014DFR30350), National Key Research and

519

Development Program of China (No.2016YFD0401202), and Youth Innovation Fund

520

of Tianjin University of Science & Technology (No.2016LG01).

521

CONFLICT OF INTEREST

522

The authors declare no competing financial interests.


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46. Zhuang, Y.; Katayama, Y.; Ueda, J.; Tanabe, S., A brief review on red to

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47. Liu, J.-M.; Liu, Y.-Y.; Zhang, D.-D.; Fang, G.-Z.; Wang, S., Synthesis of

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GdAlO3:Mn4+,Ge4+@Au

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49. Li, Y.; Zhou, S. F.; Dong, G. P.; Peng, M. Y.; Wondraczek, L.; Qiu, J. R.,

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50. Zhao, H.-X.; Yang, C.-X.; Yan, X.-P., Fabrication and bioconjugation of BIII and

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51. Chen, L.-J.; Sun, S.-K.; Wang, Y.; Yang, C.-X.; Wu, S.-Q.; Yan, X.-P.,

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53. Chen, L.-J.; Yang, C.-X.; Yan, X.-P., Liposome-coated persistent luminescence

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54. Wu, S. Q.; Yang, C. X.; Yan, X. P., A dual-functional persistently luminescent

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55. Maldiney, T.; Bessière, A.; Seguin, J.; Teston, E.; Sharma, S. K.; Viana, B.; Bos,

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in vivo activation of persistent nanophosphors for optical imaging of

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vascularization, tumours and grafted cells. Nat. Mater. 2014, 13, 418-426.

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56. Li, Y.-J.; Yan, X.-P., Synthesis of functionalized triple-doped zinc gallogermanate

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57. de Chermont, Q. l. M.; Chaneac, C.; Seguin, J.; Pelle, F.; Maitrejean, S.; Jolivet,

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Page 34 of 48

Page 35 of 48


727 728 729

Figure 1. Schematic illustration of the utilization of the antibody-ZGGO nanoprobes

730

for orally administrated in vivo bioimaging.

731

Figure 2. Structural characterization and persistent luminescence properties of the

732

ZGO and ZGGO：(A) The excitation and emission spectra of ZGGO and ZGO; (B)

733

NIR afterglow decay curve of ZGGO and ZGO powder after 120 s irradiation with a

734

254-nm UV lamp；(C) The XRD patterns of ZGO and ZGGO PLNPs powder. (D) The

735

photographs of the ZGO (left) and ZGGO (right) nanomaterials solid powder under

736

sunlight (up) and UV 254 nm (down).

737

Figure 3. HRTEM images of ZGGO (A) and ZGO (E); EDX analysis of the ZGGO

738

(B) and ZGO (F); Size distribution of ZGGO and NH2-ZGGO (C) and ZGO

739

nanoparticles; The decay curves of ZGGO (D) and ZGO (H).

740

Figure 4. Evaluation of the photostability of ZGO and ZGGO powder in the different

741

media: common biomolecules (A-B), amino acids (C-D), and metal ions (E-F).

742

Figure 5. Evaluation of the structural stability of ZGGO (A, C, E) and ZGO (B, D, F)

743

power in different simulated media.

744

Figure 6. (A) In vitro viability of 3T3, MCF-7 and HeLa cell lines incubated with

745

ZGGO of various concentrations for 24 h; (B) The weight change of the normal mice

746

with or without gavage by the ZGGO imaging probes (1 mg mL-1, 0.5 mL); (C) FT-IR

747

spectra of ZGGO, NH2-ZGGO and Antibody- ZGGO; (D) The PL intensity of ZGGO,

748

antibody-ZGGO and L. reuteri-ZGGO incubation with PBS (10 mM, pH 7.4). The 35 ACS Paragon Plus Environment

(G)


749

inset was the photograph of the dispersion of ZGGO, antibody-ZGGO and L.

750

reuteri-ZGGO incubation with ultrapure water under 254-nm UV excitation. (E) Zeta

751

potential of ZGGO, NH2-ZGGO and antibody-ZGGO; (F) The fluorescence intensity

752

change of the ZGGO against time immersed in ultrapure water.

753

Figure 7. The representative TEM images of normal L. reuteri (A-B), and normal L.

754

reuteri after incubating for 6 h with ZGGO nanoparticles (C-D).

755

Figure 8. The representative TEM images of L. reuteri competent cell after

756

incubating for 6 h with ZGGO nanoparticles (A-B), L. reuteri competent cell

757

incubating with ZGGO by electroporation treatment (C-D), and L. reuteri after

758

incubating for 4 h with the antibody-ZGGO nanoparticles (E-F).

759

Figure 9. In vitro viability of L. reuteri cell incubated with antibody-ZGGO of

760

various concentrations for 12 h and 24 h.

761

Figure 10. Bio-distribution of NH2-ZGGO and L. reuteri-ZGGO inside the mice body

762

after oral administration. (A) In vivo NIR luminescence images of PBS dispersion of

763

NH2-ZGGO (1 mg mL-1, 0.5 mL) were excited for 10 min using a LED lamp before

764

gavage. (B) In vivo NIR luminescence images of L. reuteri-ZGGO (10-9 cfu mL-1 in

765

PBS, 0.5 mL) were excited for 10 min using a LED lamp before gavage. The mice

766

were reactivated with a LED lamp at 120 min post administration; (C) The FL of

767

isolated organs of mice after 5 min irradiation with a LED lamp recorded by CCD

768

camera; (D) Ga concentration of ZGGO in isolated organs of mice measured by

769

ICP-MS elemental analysis. The acquisition was performed 24 h after the application 36 ACS Paragon Plus Environment

Page 36 of 48

Page 37 of 48


770

of ZGGO.

771



772 773

Figure 1. Schematic illustration of the utilization of the antibody-ZGGO nanoprobes

774

for orally administrated in vivo bioimaging.

775


Page 38 of 48

Page 39 of 48


776

777

Figure 2. Structural characterization and persistent luminescence properties of the

778

ZGO and ZGGO：(A) The excitation and emission spectra of ZGGO and ZGO; (B)

779

NIR afterglow decay curve of ZGGO and ZGO powder after 120 s irradiation with a

780

254-nm UV lamp；(C) The XRD patterns of ZGO and ZGGO PLNPs powder. (D) The

781

photographs of the ZGO (left) and ZGGO (right) nanomaterials solid powder under

782

sunlight (up) and UV 254 nm (down).

783



Page 40 of 48

784 785

Figure 3. HRTEM images of ZGGO (A) and ZGO (E); EDX analysis of the ZGGO

786

(B) and ZGO (F); Size distribution of ZGGO and NH2-ZGGO (C) and ZGO

787

nanoparticles; The decay curves of ZGGO (D) and ZGO (H).


(G)

Page 41 of 48


788 789

Figure 4. Evaluation of the photostability of ZGO and ZGGO powder in the different

790

media: common biomolecules (A-B), amino acids (C-D), and metal ions (E-F).

791



792 793

Figure 5. Evaluation of the structural stability of ZGGO (A, C, E) and ZGO (B, D, F)

794

power in different simulated media.

795


Page 42 of 48

Page 43 of 48


796 797

Figure 6. (A) In vitro viability of 3T3, MCF-7 and HeLa cell lines incubated with

798

ZGGO of various concentrations for 24 h; (B) The weight change of the normal mice

799

with or without gavage by the ZGGO imaging probes (1 mg mL-1, 0.5 mL); (C) FT-IR

800

spectra of ZGGO, NH2-ZGGO and Antibody- ZGGO; (D) The PL intensity of ZGGO,

801

antibody-ZGGO and L. reuteri-ZGGO incubation with PBS (10 mM, pH 7.4). The

802

inset was the photograph of the dispersion of ZGGO, antibody-ZGGO and L.

803

reuteri-ZGGO incubation with ultrapure water under 254-nm UV excitation. (E) Zeta

804

potential of ZGGO, NH2-ZGGO and antibody-ZGGO; (F) The fluorescence intensity

805

change of the ZGGO against time immersed in ultrapure water.

806



807 808

Figure 7. The representative TEM images of normal L. reuteri (A-B), and normal L.

809

reuteri after incubating for 6 h with ZGGO nanoparticles (C-D).

810


Page 44 of 48

Page 45 of 48


811 812

Figure 8. The representative TEM images of L. reuteri competent cell after

813

incubating for 6 h with ZGGO nanoparticles (A-B), L. reuteri competent cell

814

incubating with ZGGO by electroporation treatment (C-D), and L. reuteri after

815

incubating for 4 h with the antibody-ZGGO nanoparticles (E-F).

816



817 818

Figure 9. In vitro viability of L. reuteri cell incubated with antibody-ZGGO of

819

various concentrations for 12 h and 24 h.


Page 46 of 48

Page 47 of 48


820

821 822

Figure 10. Bio-distribution of NH2-ZGGO and L. reuteri-ZGGO inside the mice body

823

after oral administration. (A) In vivo NIR luminescence images of PBS dispersion of

824

NH2-ZGGO (1 mg mL-1, 0.5 mL) were excited for 10 min using a LED lamp before

825

gavage. (B) In vivo NIR luminescence images of L. reuteri-ZGGO (10-9 cfu mL-1 in

826

PBS, 0.5 mL) were excited for 10 min using a LED lamp before gavage. The mice

827

were reactivated with a LED lamp at 120 min post administration; (C) The FL of

828

isolated organs of mice after 5 min irradiation with a LED lamp recorded by CCD

829

camera; (D) Ga concentration of ZGGO in isolated organs of mice measured by

830

ICP-MS elemental analysis. The acquisition was performed 24 h after the application

831

of ZGGO. 47 ACS Paragon Plus Environment


832

TOC Graphic

833 834

A Proof-of-concept study: Applying of persistent luminescence nanophosphor based

835

long-term bioimaging for investigation of food-borne probiotics bio-distribution in

836

vivo.


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Persistent Luminescence Nanophosphor Involved Near-Infrared

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